Jaroslaw Dzbek

Bernard Korzeniewski

Control over action potential, calcium peak and average fluxes in cyclic quasi-steady-state ion transport system in cardiac myocytes: in silico studies

Keywords: Metabolic Control Analysis, heart electrophysiology, ion channel, ion pump, action potential, calcium signalling cation channel, Kr: delayed rectifier channel, rapid component, CaL delayed rectifier channel, slow component, CaT: T-type Ca 2+ channel, Cab: sarcolemmal

published or not. The documents may come

Control over action potential, calcium peak and average fluxes in the cyclic quasi-steady-state ion transport system in cardiac myocytes: in silico studies Jaroslaw Dzbek, Bernard Korzeniewski

Introduction

Metabolic Control Analysis (MCA) [START_REF] Kacser | The control of flux[END_REF][START_REF] Heinrich | A linear steady-state treatment of enzymatic chains. General properties, control and effector strength[END_REF][START_REF] Fell | Metabolic control analysis: a survey of its theoretical and experimental development[END_REF] has appeared to be an immensely useful quantitative tool for analysing the dynamic behaviour of biochemical systems. It was used in a great number of experimental and theoretical studies concerning the control of metabolic pathways (see [START_REF] Rais | Control of threonine biosynthesis in E. coli[END_REF][START_REF] Brown | Control analysis applied to the whole body: Control by body organs over plasma concentrations and organ fluxes of substances in the blood[END_REF][START_REF] Groen | Quantification of the contribution of various steps to the control of mitochondrial respiration[END_REF][START_REF] Jeneson | A metabolic control analysis of kinetic controls in ATP free energy metabolism in contracting skeletal muscle[END_REF][START_REF] Wisniewski | Distribution of flux control among the enzymes of mitochondrial oxidative phosphorylation in calcium-activated saponin-skinned rat musculus soleus fibers[END_REF][START_REF] Korzeniewski | Theoretical studies on the control of oxidative phosphorylation in muscle mitochondria: Application to mitochondrial deficiencies[END_REF] just for a few examples). In its original form MCA concerns steady-state system properties. Its most fundamental idea is based on the ratio of the relative change (dY/Y) in some variable Y caused by a small relative change (dX/X) in some parameter/variable X, to the latter change:

X X Y Y ∂ ∂ ( 1 )
In principle these changes are infinitesimal, but in practice sufficiently small finite changes can be considered. Several coefficients have been defined within MCA. Perhaps the most important and most frequently used is the flux control coefficient (FCC) expressed as a relative change in the flux J through a given system divided by a relative change in a given enzyme concentration/activity E i that caused the change in the flux:

i i J Ei E E J J C ∂ ∂ = ( 2 )
The flux control coefficient for a given enzyme is a global parameter: its value depends on the kinetic properties of the whole system and not only on the properties of this enzyme. The values of FCCs can be different in different steady states. Usually, in linear pathways, the value of the flux control coefficient of a given enzyme is between 0 (no control over flux) and 1 (entire control over flux). It was demonstrated that in linear metabolic pathways the values of FCCs for all components of the system sum up to unity (this is the so-called summation property):

1 1 = ∑ = n i J Ei C ( 3 )
However, this property does not (have to) concern branched pathways. Metabolic Control Analysis demonstrated that the control over the flux is distributed among many different steps and therefore there are no rate-limiting steps. Flux control coefficients may be attributed not only to (single) enzymes, but also to metabolic blocks (groups of enzymes), carriers (e.g. ATP/ADP carrier) and physical processes (e.g. proton leak through the inner mitochondrial membrane).

Another important coefficient is the concentration control coefficient (CCC) defined as the ratio of the relative change in a given metabolite concentration M j to the relative change in a given enzyme concentration/activity E i that caused the change in the metabolite concentration: The values of MCC can be either positive or negative. In a given metabolic system the sum of the values of all concentration control coefficients equals zero (this is the so-called connectivity property):

0 1 = ∑ = n j Mj Ei C ( 5 )
Yet another coefficient defined within MCA is the elasticity coefficient expressing the sensitivity of a given enzymatic reaction rate v k to the concentration of a given metabolite M j :

j j k k k Mj M M v v e ∂ ∂ = ( 6 )
Generally the values of flux control coefficients of particular components of some system are inversely proportional to the values of their elasticity coefficients. Some other coefficients, for instance the response coefficient, have been defined within MCA. However, they are not directly related to the present theoretical study.

As it was mentioned above, Metabolic Control Analysis is traditionally applied to steady-states. It also was shown, that MCA framework is not straightforwardly applicable to transient and oscillatory systems [START_REF] Reijenga | Control analysis for autonomously oscillating biochemical networks[END_REF][START_REF] Ingalls | Sensitivity analysis of stoichiometric networks: an extension of metabolic control analysis to non-steady state trajectories[END_REF][START_REF] Kholodenko | Control analysis of periodic phenomena in biological systems[END_REF][START_REF] Demin | Control analysis of stationary forced oscillations[END_REF]. However, several theoretical approaches have been defined in order to tackle problems arising in the analysis of such systems [START_REF] Reijenga | Control analysis for autonomously oscillating biochemical networks[END_REF][START_REF] Ingalls | Sensitivity analysis of stoichiometric networks: an extension of metabolic control analysis to non-steady state trajectories[END_REF][START_REF] Kholodenko | Control analysis of periodic phenomena in biological systems[END_REF][START_REF] Demin | Control analysis of stationary forced oscillations[END_REF][START_REF] Conradie | Summation theorems for flux and concentration control coefficients of dynamic systems[END_REF]. It has been demonstrated that in quasi-steady-state systems with a constant oscillation period (imposed through e.g. external stimulation) it is possible to define variables whose unique relative changes can be calculated and analyzed under the framework of MCA [START_REF] Kholodenko | Control analysis of periodic phenomena in biological systems[END_REF][START_REF] Demin | Control analysis of stationary forced oscillations[END_REF]. In such systems, variable values return periodically to the initial value, average fluxes during one cycle can be determined and such variables as the amplitude of, duration of, and area under the oscillation (pulse) of some metabolite concentration (or thermodynamic force) can be defined.

A good example of such a cyclic quasi-steady-state system is the ion circulation and action potential generation in isolated cardiac myocytes. In this system heart cells are periodically stimulated electrically with a constant stimulation interval. This stimulation increases transiently some ion channel conductivities, generates an ion movement across the cell membrane and sarcoplasmatic reticulum membrane, and thus induces changes in the membrane potential. Several kinetic models of this system have been developed [START_REF] Difrancesco | A model of cardiac electrical activity incorporating ionic pumps and concentration changes[END_REF][START_REF] Luo | A dynamic model of the cardiac ventricullar action potential: I. simulations of ionic currents and concentration changes[END_REF][START_REF] Matsuoka | Role of individual ionic current systems in ventricular cells hypothesized by a model study[END_REF] that are generally similar, although differ in details. The elements of the system taken into account explicitly in the model developed by Matsuoka and co-workers [START_REF] Matsuoka | Role of individual ionic current systems in ventricular cells hypothesized by a model study[END_REF], used in the present study, are presented in Fig. 1. The system is composed of several pumps and ion channels that conduct Na + , K + and/or Ca 2+ currents.

The most important events occurring in the system during one oscillation period are as follows.

External electrical stimulation causes a very high, but very short activation of Na channel, what leads to an intensive short-term inward sodium current I Na . This is related to a sudden increase in the membrane potential from about -85 mV (resting potential) to over +40 mV (action potential). As a result, K1 channel closes, allowing to generate a plateau phase in the membrane potential. Increase of the membrane potential above -30 mV activates CaL channel and causes an influx of Ca 2+ ions from the extracellular space to cytosol (I CaL ). Increased Ca 2+ concentration promotes calcium-induced calcium release (CICR) Biochemical Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society from sarcoplasmic reticulum (SR). This flux occurs through RyR channels (I RyR current) with its amplitude being about two orders of magnitude higher than that of the I CaL ; nevertheless RyR channels are quickly deactivated and the ratio of total Ca 2+ entry through RyR and CaL channels is 15:1. The high membrane potential causes also a slow, time-dependent activation of K (Ks + Kr) channels and an increase in K + ion efflux from the cell (I Ks + I Kr ). As a result the membrane potential decreases, K1 channel is activated and the decrease in the membrane potential is further accelerated. Finally, the ATP-driven Na + /K + pump (NaK) and Na + /Ca 2+ exchanger (NaCa) bring the system back to the initial resting state. The changes in the membrane potential result from the ion (charge) movement across the cellular membrane and their magnitude is dependent on the capacitance of the membrane. 400 ms after a previous external stimulation the next stimulation is applied and the whole cycle is repeated. Fig. 2 shows the simulated time course of the membrane potential and cytosolic Ca 2+ concentration during one cycle. Of course, the calcium pulse is crucial for the stimulation of the myocyte contraction (driven by actomyosin-ATPase) and at least some components of the ATP-producing system. The above description of the behaviour of the system is a generalized one and the detailed description can be found in [START_REF] Difrancesco | A model of cardiac electrical activity incorporating ionic pumps and concentration changes[END_REF][START_REF] Luo | A dynamic model of the cardiac ventricullar action potential: I. simulations of ionic currents and concentration changes[END_REF][START_REF] Matsuoka | Role of individual ionic current systems in ventricular cells hypothesized by a model study[END_REF].

In the present theoretical study the metabolic control exerted by particular components of the ion transport system in cardiac myocytes (channels, pumps, exchangers) over the amplitude and duration of the action potential, area under the calcium peak and average fluxes through particular components is analyzed. It is found that: 1. the control is distributed between different steps; 2. some steps tend to have more control than other steps; and 3. different steps have the greatest control over different variables.

Generally, it is demonstrated that Metabolic Control Analysis is very useful in describing not only steadystates, but also cyclic quasi-steady-states in biochemical/biophysical systems.

Biochemical

Theoretical procedures

In the computer simulations performed in the present study the model of ion transport in isolated cardiac myocytes developed previously by Matsuoka and co-workers [START_REF] Matsuoka | Role of individual ionic current systems in ventricular cells hypothesized by a model study[END_REF] was used without any modifications. Within this model the dependences of the activities of particular elements of the system (channels, pumps, exchangers) on ion concentrations, membrane potential and time are expressed in the form of kinetic equations. The changes of ion concentrations and membrane potential over time are described as a set of ordinary differential equations. The authors of the model validated it successfully by a comparison of computer simulations with experimental data concerning the action potential time course, internal Ca 2+ concentration transient and sarcomere length changes occurring during the contraction event. They studied in the theoretical way the effect of large-scale changes in some selected channel activities on the shape of the action potential and the magnitude of some selected currents; however, they did not perform the control analysis of the system and did not deal with the variables analyzed in the present study.

The discussed model was implemented by us in the Fortran programming language. In order to check that no errors were made during the implementation several system properties, including those presented in Fig. 2, were simulated and compared with the simulations presented in the original paper [START_REF] Matsuoka | Role of individual ionic current systems in ventricular cells hypothesized by a model study[END_REF]. The complete description of the model is also given in the above publication.

The control coefficient of particular steps X i (channels, pumps, exchangers) over different variables Y j (amplitude and duration of the action potential, area under the Ca 2+ peak, average fluxes through particular channels) were calculated using the following equation:

i i j j Yj Xi X X Y Y C Δ Δ = ( 7 )
In subsequent computer simulations the original rate constants of particular steps were increased by a relative factor of 0.01 (by 1 %) and the relative changes in the variable Y j between the original and the new quasi-steady-state were recorded. In subsequent series of variable estimation Y represented: A. action potential amplitude (the maximal difference, in mV, between the peak action potential and the resting potential: compare Fig. 2); B. action potential duration (apd50) (width of the action potential peak, in ms, at 50 % of the maximal difference between the action potential and the resting potential: compare Fig. 2) representing the actual time span of electric excitation during each cycle; C. area under the Ca 2+ pulse (the integral of Ca 2+ concentration over time after subtracting the resting Ca 2+ concentration) generally representing the potential magnitude of influence on Ca 2+ sensitive subsystems (see Fig. 2); D. average fluxes (the net amounts of translocated charges related to some particular ion divided by the oscillation period, in pA) through selected elements of the system during the whole pulse period (400 ms), which quantitatively represent the contribution of particular elements to the overall ion-cycling activity of the system. The first three of the above mentioned variables are often easily assessed during a typical cell behaviour recording experiment.

Biochemical Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society

Theoretical results

The ion circulation system in cardiac myocytes presented in Fig. 1 is an extremely 'branched' system in the sense that there are many independent ways in which particular ions can be transported across the plasma membrane and sarcoplasmic reticulum membrane. Additionally, apart from two exchangers, namely the ATP-driven Na + /K + pump (NaK) and Na + /Ca 2+ exchanger (NaCa), the transport of Na + , K + and Ca 2+ ions is independent. Therefore, different channels affect each other only via a common ion transported and, indirectly, via the changes in the membrane potential related to the ion redistribution across the plasma membrane. For this reason it is very important to know the values of the ionic currents through particular channels. Fig. 3 presents the simulated average (in pA) currents (fluxes) during one cycle (400 ms). The ion flow through particular channels is either exclusively or predominantly unidirectional during the cycle (not shown, see also [START_REF] Matsuoka | Role of individual ionic current systems in ventricular cells hypothesized by a model study[END_REF]), and therefore the net ion movement is (almost) identical with the total ion movement. It can be seen that the greatest currents are conducted by the sarcoplasmic reticulum pump and channels: SRU, SRL, RyR and SRT. From among the plasma membrane channels, the greatest K + currents are conducted by NaK and K1 channels, the greatest Na + currents are conducted by NaK and bNSC channels, while the greatest Ca 2+ currents are conducted by NaCa and CaL channels. One can expect that these channels play an important role in determining the dynamic properties of the system.

Fig. 4 presents the simulated control coefficients of particular elements of the system over the action potential amplitude. It can be seen that an increase in the activity of the ATP-driven Na + /K + pump (NaK) increases this amplitude, while an activation of Na channel, bNSC channel and CaL channel moderately decreases the amplitude. The activities of other channels have a much smaller impact on the magnitude of the action potential amplitude. Some channels, namely Ks, Kr, CaT, Cab, to, Kpl, l(Ca) and KATP, have apparently no control.

Fig. 5 shows the control exerted by particular channels on the duration of the action potential (peak width at 50 % of the action potential amplitude). One can see that the pattern of control is significantly different here than in the case of the action potential amplitude. bNSC, K1 and Na channels have a strong negative control, while NaK pump exerts a moderate positive control over the duration of the action potential. Moreover, the absolute values of control coefficients are generally greater here than in the previous case (the action potential duration is more sensitive to the channel activities than the action potential magnitude).

The simulated control coefficients of particular channels over the area under the Ca 2+ peak are presented in Fig. It also has to be noted that these control coefficient values are much greater than in the previous two cases, meaning that the behaviour of the calcium handling subsystem is much more sensitive to the changes in the activities of the ion transporting elements than the action potential generation subsystem.

Discussion

The present paper analyses the control exerted by particular components (channels, pumps, exchangers) of the cyclic quasi-steady-state ion transport system in isolated cardiac myocytes over several variables including the amplitude and duration of the action potential, the area under the calcium transient and average fluxes through particular channels during one oscillation period. It is demonstrated that, first, it is possible to determine the pattern of metabolic control not only in a steady-state system, but also in a quasi-steady-state system with periodic changes of fluxes, metabolite concentrations and thermodynamic forces. Second, the control is distributed among many (essentially all) components of the system, like in the majority of the systems analysed using Metabolic Control Analysis. Third, different variables are controlled to different extent by different components of the system. K + , Na + and Ca 2+ are transported by several alternative ways: channels, pumps and exchangers. However, as it can be seen in Fig. 3, the average fluxes during one cycle through these ways can be very different for a given ion. In some cases the transport of two ions is coupled and has a fixed stoichiometry (NaK pump, NaCa exchanger), and in some cases an independent (without a fixed stoichiometry) flow of two or three ions through one channel occurs (CaL, bNSC, l(Ca), to, Ks, Na). Of course, one can suppose that the channels with the highest average fluxes play the most important role in the system, although the time variations of the fluxes during one oscillation period are also of a great importance, especially for the shape of the action potential and calcium transient.

The amplitude of the action potential is controlled to the greatest extent (Fig. 4) by NaK pump, the element of the system that conducts the highest K + and Na + fluxes. Also bNSC and Na channels that conduct the second and third greatest Na + currents, respectively, exert a significant control over the action potential amplitude. Interestingly, the action potential amplitude is mainly modified via changing the resting potential value and not the peak value of the action potential. This is why the outward sodium currents (hyperpolarizing currents) have a positive control over the amplitude, while the inward sodium currents have a negative control over it.

The negative control over the action potential duration is mostly exerted (Fig. 5) by bNSC, K1 and Na channels and the positive control by NaK channel. All these channels conduct significant K + and/or Na + fluxes. A negative control seems to be associated with the Na + inward and K + outward fluxes, while a positive control with the opposite fluxes.

The area under the Ca 2+ peak is significantly increased by an increased activity of bNSC, CaL and Na channels and by a decreased activity of NaK and NaCa exchangers. CaL channel conducts an influx of Ca 2+ ions from the extracellular space to the cytosol, while the NaCa exchanger conducts the opposite Ca 2+ current, and therefore the effect of their activity on the calcium peak size is straightforward. bNSC and Na channels transport Na + ions to the cytosol, while NaK pump transports these ions outside the cell.

Because the outward Ca 2+ transport by NaCa is coupled with a Na + influx to the cytosol, a high external and low internal [Na + ] promotes the outward Ca 2+ transport and therefore diminishes the calcium peak.

Interestingly, although the SR-related calcium channels conduct the highest fluxes (see Fig. 4), their

Biochemical Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755

© 2007 Biochemical Society control over the area under the Ca 2+ peak is very moderate. This is mostly caused by the fact that the release of Ca 2+ from SR to the cytosol through RyR and SRL and the ATP-driven Ca 2+ uptake by SRU are significantly controlled by the cytosolic Ca 2+ itself: as it was discussed above high sensitivities to metabolite concentrations (high elasticity coefficients) are associated with low a control over system variables.

The activities of four channels, namely Na, NaK, K1 and bNSC, control to a significant extent the values of the variables analyzed above: the action potential amplitude, action potential duration and area under the calcium peak. Therefore, it is interesting to test what controls the fluxes just through these channels. The simulated flux control coefficients of all components of the system over the currents through Na, NaK, K1 and bNSC, presented in Fig. 7, demonstrate that the control is distributed mostly among these channels themselves. They are elements of the Na + and K + transport system, but not the Ca 2+

transport system. This suggests that the Na + and K + transport system is kinetically superior in relation to the Ca 2+ transport system: the former controls the latter via the membrane potential and Na + gradient across the plasma membrane (driving the NaCa exchanger). In fact, the system composed of only these four channels is able to generate the shape of the membrane potential pulse similar to the original one, shown in Fig. 2, although, of course, it can not generate the original Ca 2+ pulse (simulations not shown).

The simplistic formulation of the adenosinephophates conversion used in the model makes the concentrations of ATP, ADP and P i almost constant during the simulated oscillation cycle. However, in reality, some important changes in these concentrations may take place. Therefore, a more sophisticated ATP production block might also contribute to the distribution of the control over the system variables.

Eight channels of eighteen, namely Ks, Kr, CaT, Cab, to, Kpl, l(Ca) and KATP, seem to have essentially no control over the analyzed system variables: the amplitude and duration of the action potential, area under Ca 2+ peak and average fluxes through particular channels. This fact seems to be strictly associated with the very low currents conducted by these channels (compare Fig. 3). In fact in the simulation in which these channels were eliminated from the system (their activity was assumed to be zero), the general behaviour of the system remained essentially unchanged (simulations not shown).

Therefore a question arises, why these channels did appear during the biological evolution and still exist in cardiac myocytes. It is possible that they play some unknown function and/or are activated by some unknown factors in stressing conditions, for instance during an energetic crisis and/or hypoxia. For example, the current conveyed by KATP channel becomes influential only under a heavy cytosolic ATP depletion to the level of 0.5 mM -in such conditions the channel activity increases 100 times when compared to its basic activity under typical 5 mM ATP. It is also interesting that some of these channels (namely Kr, CaT, Cab, KATP and Kpl) tend to control only their own activities with the values of The ion transport system analyzed in the present study is in many aspects very different from a 'typical' metabolic biochemical pathway (leaving apart the fact that it is a cyclic, quasi-steady-state system). First, there are essentially no (or, at best, very few) linear sequential reaction chains here.

Second, the system is extremely 'branched' -many different ways of the flow of a given ion through the plasma membrane or sarcoplasmic reticulum membrane are present. Third, there is no chemical conversion of one metabolite into another (if ATP hydrolysis by NaK and SRU is ignored), but only a purely physical flow of different ions from one compartment into another. This architecture of the system has some important dynamic consequences. First, while the channels conducting a common ion can 'communicate' through this ion concentration and the membrane electrical potential, then the channels not conducting common ions can exert an influence on each other only via this potential (in a sense, the membrane potential is a 'common metabolite' for all channels in a given membrane). Second, the summation property can not be applied to the system -the flux control coefficients of all components over particular (average) fluxes do not sum up to unity. Third, the currents of positively-charged ions in one direction force the currents of positively-charged ions in the opposite direction (this is why the system is cyclic and not unidirectional; however, one must bear in mind that the cyclic ion movement is driven by an unidirectional ATP hydrolysis by NaK and SRU).

The presented coefficients of the control over the action potential amplitude, action potential duration and area under the calcium peak exhibit some resemblance to the concentration control coefficients: for instance, they can be either positive or negative and are related to metabolite concentrations and/or thermodynamic potentials. On the other hand, they refer to quasi-steady-state properties of the system and do not fulfill the connectivity property. Therefore, they are certainly not identical with the concentration control coefficients.

One must also bear in mind that the evaluated coefficients represent only a single point in the parameter (activities) space. Thus, a significant change in some activities will usually redefine the values of different control coefficients. This means, that under non-standard conditions the control over different parameters may be shifted to other components of the system. Such a situation may take place, for example, when some genes coding particular ion channels are knocked out or overexpressed or when a permanent inhibition of particular enzymes occurs.

In the current paper we present only a few system variables selected for control analysis; in our simulations, however, several other variables were considered as well, for instance the area under the action potential, calcium peak amplitude or ATP usage by NaK and SRU, and many more (data not shown). In fact, any conceivable, arbitrary variable describing a particular property of the quasi-steadystate system in mind can be used as a target of the control by the dynamic elements of the entity. Of The knowledge of the control of particular elements of the system over different system variables gives a better insight into the functioning of the system and therefore is important for the pure science. It has also very important potential practical applications [18], possibly in inventing treatment for heart diseases, for instance those related to the heart beating cycle. The model predictions can be potentially tested by analyzing, in the experimental way, the effect of different mutations leading to small changes in particular channel activities or concentrations on different system variables. They may also be validated by using selective inhibitors of particular ion transporters in experiments recording selected system variables.

Summing up, in the present study the control analysis of chosen variables, namely the action potential amplitude, action potential duration, area under Ca 2+ peak and average fluxes through particular ion channels in the cyclic ion circulation system in isolated cardiac myocytes was performed. It is shown that: 1. the control is distributed among different components of the system (channels, exchangers, pumps); 2. different variables are controlled by different components to a different extent; 3. the Na + and K + transport system exerts a significant control over the Ca 2+ transport system, but not inversely.

Generally, it is practically demonstrated that the approach based on Metabolic Control Analysis can be very useful in analyzing not only steady-state, but also cyclic quasi-steady-state systems. Fig. 3. Average currents (net amounts of translocated charges related to some particular ion divided by the duration of the oscillation period, in pA) through particular channels during one cycle period (400 ms).

I X , ion current through system element X (see Abbreviations for full ion transporter names). Na + , K + , Ca 2+ : currents of Na + , K + and Ca 2+ ions through the cellular membrane; SR Ca 2+ : currents of Ca 2+ ions through the sarcoplasmic reticulum membrane. For the transporters that conduct different ions particular ion currents are presented separately. Note the alternative scale for the SR (sarcoplasmic reticulum) currents.

Fig. 4. Simulated control coefficients of particular system elements over the action potential amplitude.

The control coefficients were calculated using Equ. 7, where X represents a given system element activity and Y represents the action potential amplitude (compare Fig. 2). See Abbreviations for full ion transporter names.

Fig. 5. Simulated control coefficients of particular system elements over the action potential duration at 50% height. The control coefficients were calculated using Equ. 7, where X represents a given system element activity and Y represents the action potential duration (compare Fig. 2). See Abbreviations for full ion transporter names. Fig. 6. Simulated control coefficients of particular system elements over the area under Ca 2+ transient.

The control coefficients were calculated using Equ. 7, where X represents a given system element activity and Y represents the area under Ca 2+ transient (compare Fig. 2). See Abbreviations for full ion transporter names.

Fig. 7

 7 Fig.7presents the simulated flux control coefficients of particular elements of the system quantifying the control over average ion currents exerted by the ion transporters that have the most significant control over the action potential amplitude and duration, and over the calcium pulse (Na, NaK, K1 and bNSC) and, not surprisingly, also have high values of ion fluxes. It can be seen that the control over the ion fluxes through these channels is distributed mostly among just these channels. The only major exception is the relatively high control of CaL channel over the flux through the NaK channel.

1 .C

 1 This is contrary to the self control coefficients values for the significant channels, for which the changes in their respective activities are compensated, thus leading to only a minor change in fluxes ( values equal to or smaller than 0.2).

  course, the selected variables should hold a potentially useful biological and/or physical meaning. The purpose of the present study was to analyze only some representative examples. Here, we are showing the plausibility and usefulness of the evaluation of control coefficients over arbitrarily defined parameters of systems oscillating with a constant period, thus enabling a quantitative analysis of interdependencies Biochemical Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Societybetween the elements of the entity. However, our approach does not deal with the changes in the frequencies of oscillations; in fact, the oscillation period is defined as a constant in the employed model.Of course, in the heart in vivo the cardiac cells are not excited by an artificial electrical stimulation, but by neural signals coming from the sinoatrial (SA) node. The frequency of this excitation may vary depending on conditions, for instance on the presence of different hormones or neural signals coming from the brain. However, the discussed excitation is still external and hierarchically superior in relation to the ion circulation system taken into account within the model (additionally, the model does not involve any feedback signals from cardiac myocytes to the pacemaker cells in the SA node). Therefore, the model can not be used to study the control of different system elements over the frequency of the excitation and contraction events.

Fig. 1 .Fig. 2 .

 12 Fig.1. Scheme of the ion transport system in cardiac myocytes. Particular channels, exchangers and pumps participating in the Na + , K + and Ca 2+ transport across the plasma membrane and sarcoplasmic reticulum membrane are shown (see Abbreviations for the full names of the transporters). The numbers at the arrows indicate stoichiometries; X o , X i , ion X in the extracellular and cytosolic compartments, respectively. Note that several channels/exchangers/pumps conduct more than one ion type.

Fig. 7 .

 7 Fig.7. Simulated flux control coefficients of particular channels over average currents (net amounts of translocated charges related to some particular ion divided by the oscillation period) through four selected

Fig 2 .

 2 Fig 2.

  Fig 3.

Fig 4 .

 4 Fig 4.

Biochemical Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society

  [START_REF] Groen | Quantification of the contribution of various steps to the control of mitochondrial respiration[END_REF]. Again, the control is distributed among several steps, from among which the NaK pump exerts a strong negative control, the bNSC exchanger and CaL channel exert a moderate positive control, while the control kept by other channels is significantly smaller. And again, several channels,

namely Ks, Kr, Kpl, l(Ca), to, KATP, Cab and CaT seem to keep essentially no control.

Biochemical Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society

Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society

	Biochemical Fig 5. Biochemical
		0.2
	control coefficients control coefficients	-0.2 -0.1 0 0.1	05
		-0.3
			Na	Na NaK	NaK K1	K1 bNSC	bNSC Kr	Kr CaL	CaL NaCa	NaCa Ks	Ks CaT	CaT Cab	Cab KATP	KATP Kpl	Kpl l(Ca)	l(Ca) to	to RyR	RyR SRU	SRU SRT	SRT SRL	SRL

Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society

	Biochemical
	Fig 6.
		2
	control coefficients	-2 -1 0 1
		-3
		Na	NaK	K1	bNSC	Kr	CaL	NaCa	Ks	CaT	Cab	KATP	Kpl	l(Ca)	to	RyR	SRU	SRT	SRL

Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society

	Biochemical
	Fig 7.
		0.4
		0.2																	I Na
		0
		0.4
		0.2																	I NaK
	control coefficients	0.4 0
		0.2																	I K1
		0
		0.4
		0.2																	I bN SC
		0
		Na	NaK	K1	bNSC	Kr	CaL	NaCa	Ks	CaT	Cab	KATP	Kpl	l(Ca)	to	RyR	SRU	SRT	SRL

Journal Immediate Publication. Published on 6 Feb 2007 as manuscript BJ20061755 © 2007 Biochemical Society


Lingli Li

Trias Asteriou

Berit Bernert

Carl-Henrik Heldin

Paraskevi Heldin
email: paraskevi.heldin@imbim.uu.se

Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: Importance of hyaluronan for the mitogenic response of PDGF-BB

Keywords: Hyaluronan synthase, hyaluronidase, growth factors, signal transduction

The glycosaminoglycan hyaluronan is important in many tissue repair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to platelet-derived growth factor (PDGF)-BB and other growth factors.

Human dermal fibroblasts exhibited the highest hyaluronan synthesizing activity in response to PDGF-BB. Analysis of hyaluronan synthase (HAS) and hyaluronidase (HYAL) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK1/2 (U0126) and for PI3 kinase (LY294002), as well as the SN50 inhibitor, that prevents translocation of the active NFkB to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signaling pathways were without any significant effect. Transforming growth factor-ß1 (TGF-ß1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Importantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hermes-1, inhibited PDGF-BB-stimulated [ 3 H]thymidine incorporation of dermal fibroblasts. We conclude that the Erk MAP kinase and PI3 kinase signaling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.

INTRODUCTION

Hyaluronan is a glycosaminoglycan consisting of repeating disaccharide units of Dglucuronic acid and N-acetyl-D-glucosamine. It is found in particular in soft connective tissues and is the major component in synovial joint fluid, vitreous body and skin, both in dermis and epidermis, contributing both to the structural and functional integrity [START_REF] Laurent | Hyaluronan[END_REF][START_REF] Tammi | Hyaluronan in the epidermis[END_REF].

Hyaluronan is synthesized by three hyaluronan synthesizing enzymes (HAS1, 2 and 3) located at the plasma membrane, and the newly synthesized molecule is extruded into the extracellular space while the synthesis is in progress [START_REF] Prehm | Hyaluronate is synthesized at plasma membranes[END_REF][START_REF] Weigel | Hyaluronan synthases[END_REF][START_REF] Tammi | Hyaluronan and homeostasis: a balancing act[END_REF]. Among the three HAS isoforms, the HAS2 isoform is required for embryonic development since mice deficient in the HAS2 gene die at embryonic day E 9.5; in contrast, mice deficient in HAS1 and HAS3 genes are viable [START_REF] Camenisch | Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronanmediated transformation of epithelium to mesenchyme[END_REF].

Hyaluronan accumulation is a prominent feature of rapidly remodeling tissues. For example, during embryonic development, hyaluronan production facilitates the migration of differentiating cells and stimulates their transformation to a mesenchymal phenotype [START_REF] Camenisch | Disruption of hyaluronan synthase-2 abrogates normal cardiac morphogenesis and hyaluronanmediated transformation of epithelium to mesenchyme[END_REF]. Furthermore, hyaluronan is involved in the skin wound-healing process from the early inflammatory process to re-epithelization and remodeling [START_REF] Chen | Functions of hyaluronan in wound repair[END_REF], and is involved in scarless fetal healing most likely by reducing collagen deposition [START_REF] Chen | Functions of hyaluronan in wound repair[END_REF][START_REF] Li | Irradiation-induced expression of hyaluronan (HA) synthase 2 and hyaluronidase 2 genes in rat lung tissue accompanies active turnover of HA and induction of types I and III collagen gene expression[END_REF]. In addition, hyaluronan overproduction promotes tumorigenesis in several tumor types, whereas suppression of hyaluronan production decreases tumorigenicity [START_REF] Jacobson | Expression of hyaluronan synthase 2 or hyaluronidase 1 differentially affect the growth rate of transplantable colon carcinoma cell tumors[END_REF][START_REF] Simpson | Inhibition of prostate tumor cell hyaluronan synthesis impairs subcutaneous growth and vascularization in immunocompromised mice[END_REF]. Accumulation of hyaluronan synthesis can arise from both increased synthesis and decreased degradation. Hyaluronan is degraded by the action of hyaluronidases (HYALs), including HYAL1, HYAL2 and PH-20 that have demonstrated bona fide hyaluronidase activity [START_REF] Stern | Devising a pathway for hyaluronan catabolism: are we there yet?[END_REF]. Hyaluronan degradation occurs locally in several tissues, for example in dermis where hyaluronidase activities have been demonstrated [START_REF] Ohno | Expression and activity of hyaluronidase in human periodontal ligament fibroblasts[END_REF]13], however, the mechanisms of hyaluronan metabolism in the connective tissues under physiological and inflammatory conditions are still largely unknown.

Hyaluronan interacts with several cell surface receptors, such as CD44, RHAMM and LYVE-1, and extracellular components, such as versican and aggrecan [START_REF] Turley | Signaling properties of hyaluronan receptors[END_REF][START_REF] Day | Hyaluronan-binding proteins: tying up the giant[END_REF]; through its complex interactions, hyaluronan affects a variety of physiological cellular events, such as cell proliferation, differentiation and migration. Because of its hygroscopic and viscoelastic properties, hyaluronan also affects cellular behavior via remodeling of the macro-and microenviroments around cells [START_REF] Heldin | Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells[END_REF][START_REF] Heldin | Chondroitin sulfate proteoglycan modulates the permeability of hyaluronan-containing coats around normal human mesothelial cells[END_REF][18][19].

Growth factors play central roles during both normal and pathological conditions, including embryogenesis, wound healing and tumor invasion, i.e. biological processes characterized by increased amounts of hyaluronan. Platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-ß1 (TGF-ß1), which are produced by both normal and transformed cells, are powerful stimulators of hyaluronan production by certain mesenchymal cells [START_REF] Heldin | Synthesis and assembly of the hyaluronan-containing coats around normal human mesothelial cells[END_REF][START_REF] Heldin | Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts[END_REF][START_REF] Yu | Both platelet-derived growth factor receptor (PDGFR)-alpha and PDGFR-beta promote murine fibroblast cell migration[END_REF][START_REF] Stuhlmeier | Differential effect of transforming growth factor beta (TGF-beta) on the genes encoding hyaluronan synthases and utilization of the p38 MAPK pathway in TGF-beta-induced hyaluronan synthase 1 activation[END_REF]. PDGF-BB mediates its cellular effects through activation of a-and ßtyrosine kinase receptors. Ligand binding causes receptor dimerization, leading to autophosphorylation of specific tyrosine residues in the intracellular parts of the receptors. This creates docking sites for SH2 domain containing signaling molecules, whereby several signaling pathways are activated [START_REF] Heldin | Mechanism of action and in vivo role of platelet-derived growth factor[END_REF]. PDGF receptors can act synergistically with a v ß 3 integrin to promote tumor cell tissue infiltration [START_REF] Woodard | The synergistic activity of alphavbeta3 integrin and PDGF receptor increases cell migration[END_REF]. However, recent studies in our laboratory on primary cultures of human dermal fibroblasts, revealed that high concentrations of exogenous hyaluronan negatively affects PDGF ß-receptor activation and cell migration in a CD44-dependent manner [25]. In normal fibroblast cultures, the intracellular signaling pathways that regulate the turnover of hyaluronan in response to external stimuli have not yet been clarified. Here, we demonstrate that the effect of PDGF-BB on hyaluronan synthesis in human dermal fibroblast cultures is due to induction of HAS2. In contrast, TGF-ß1 has no effect on hyaluronan synthesis but enhances hyaluronan degradation by stimulation of hyaluronidase activity. Moreover, we show that the Ras-Erk MAP kinase and PI3 kinase pathways are important for PDGF-BB-stimulated hyaluronan production in these cells, and that hyaluronan binding to its receptor CD44 is important for PDGF-BB-induced cell growth. medium (grade II, 0.5 U/ml, Boehringer Mannheim / DMEM) at 4˚C, dermis and epidermis were separated, using a fine forceps. Fibroblasts from the dermal sheet explants were then cultured in complete medium (DMEM containing 10% fetal bovine serum (FBS, Biowest and antibiotics), and used between passages 6 to 10 for all experiments. Normal human epidermal keratinocytes were released from the epidermal sheets by incubation with 4 mg/ml trypsin and 4 mg/ml glucose in 0.02% EDTA at 37 o C for 1 h. After gentle pipetting, 10% FBS was added to stop the enzymatic activity, whereafter keratinocytes were washed and collected by centrifugation. Keratinocytes (60-70% confluence, passage three) were grown in EpiLife TM keratinocyte medium (Sigma, # E0151), containing Keratinocyte Medium Supplement bovine pituitary extract-free and 10% FBS.

DMEM supplemented with 10% FBS was also used in the culture of HEK 293 cells, Cos-1 cells, 3T3 cells, human foreskin fibroblasts (AG1518, AG1523; Human Mutant Cell Repository, Camden, NJ, USA), mouse embryonal fibroblasts (MEFs), the mouse epithelial cell line NMuMG (kindly provided by Anita Moren, Ludwig Institute for Cancer Research, Uppsala, Sweden; medium contained also 10 µg/ml insulin), and fibroblasts derived from keloid-like dermal scars (generously provided by Bengt Gerdin, Akademiska hospital, Uppsala, Sweden).

Polyclonal antibodies against human HAS2 and HYAL2, as well as peptide synthesis

To raise polyclonal antibodies against HAS2 and HYAL2 we chose the peptide CGRRKKGQQYDMVLD that corresponds to amino acid residues 537-549 at the C-terminal domain of HAS2 and is conserved in both human and mouse HAS2 [START_REF] Watanabe | Molecular identification of a putative human hyaluronan synthase[END_REF][START_REF] Spicer | Molecular cloning and characterization of a putative mouse hyaluronan synthase[END_REF], and the peptide PGHAPGEPQLRPVG that corresponds to amino acid residues 398-411 at the C-terminal domain of human HYAL2 [START_REF] Lepperdinger | HYAL2, a human gene expressed in many cells, encodes a lysosomal hyaluronidase with a novel type of specificity[END_REF]. The peptides were synthesized using the N-(9-fluorenyl) methoxycarbonyl strategy which resulted in a carboxy-terminal amidated domain to facilitate subsequent coupling to Keyhole Limpet Hemocyanin for rabbit immunization. Furthermore, a cysteine residue was added to PGH peptide at the C-terminal domain, in order to facilitate a subsequent coupling to a SulfoLink resin (<http://www.jbc.org/cgi/content/full/278/41//cgi/redirect-inline?ad=Pierce> Pierce), and used to affinity purify the antibodies. The peptides were purified by reversed-phase high pressure liquid chromatography and rabbits were immunized (service by EUROGENETEC, Belgium).

The antisera were used directly or after purification on a HiTrap Protein G HP column (Amersham Biosciences), according to the instructions of the manufacturer.

Determination of hyaluronan synthesis

Cells (3 x 10 4 cells/well in 12 well plates, or as indicated) were incubated in complete medium for 24 h, whereafter the medium was replaced with DMEM containing 0.1% FBS (starvation medium). Then, the quiescent cells received fresh starvation medium and were stimulated for an additional The hyaluronan content in 24 h conditioned media was quantified, essentially as described previously [START_REF] Jacobson | Expression of hyaluronan synthase 2 or hyaluronidase 1 differentially affect the growth rate of transplantable colon carcinoma cell tumors[END_REF]. This assay is based on the highly specific and irreversible capture of hyaluronan molecules in the samples with immobilized hyaluronan binding protein (HABP) domain of aggrecan, from bovine cartilage [START_REF] Tengblad | Affinity chromatography on immobilized hyaluronate and its application to the isolation of hyaluronate binding proteins from cartilage[END_REF]; proteins that bind hyaluronan with such high affinity are not present in the fibroblast cultures [START_REF] Johansson | Structure and interactions of proteoglycans in the extracellular matrix produced by cultured human fibroblasts[END_REF]. The HABP-hyaluronan complexes are then detected by biotinylated-HABP (b-HABP) and streptavidin biotinylated horseradish peroxidase (HRP) followed by a chromogenic reaction. The assay was carried out on MaxiSorb 96-well Nunc-Immuno Plates (Nunc #439454) pre-coated overnight with 1 µg HABP/ml in 50 mM carbonate buffer, pH 9.5. After three washes in PBS containing 0.5% Tween 20, and blocking in PBS supplemented with 1% BSA, the hyaluronan standards (0-100 ng/ml, Mw 0.5 -1.5x 10 6 , Genzyme Hylumed) and samples, at appropriate dilutions in blocking solution, were added and the plates were incubated for 1 h at 37˚C. Following washing, 100 µl of biotinylated HABP (b-HABP; 1 µg/ml) was added and samples were incubated for 1 h. Then, after removing excess b-HABP, the b-HABP bound to the hyaluronan trapped by the HABP-coated plates was detected by incubation for 1 h with streptavidin biotinylated HRP (Amersham Bioscience #RNP1015V), followed by the addition of 100 ml 3,3',5,5' tetramethylbenzidine substrate solution (Sigma #T4444) for 15 minutes.

Then, 50 µl of 2 M H 2 SO 4 was added and the absorbance at 450 nm was measured. The hyaluronan content in the conditioned media was calculated by comparing with a standard curve made from known concentrations of hyaluronan. It is unlikely that hyaluronan binding proteins secreted into the conditioned medium affected the result, since treatment of the conditioned medium with proteases before the assay, had no effect (unpublished data).

Detection of HYAL activity in human dermal fibroblast cultures

The activity of HYALs in fibroblast conditioned media and cell-layers harvested after 6 h from unstimulated and PDGF-BB-or TGF-ß-stimulated cells was quantitatively estimated using a microtiter-based assay essentially as described by Frost and Stern [START_REF] Frost | A microtiter-based assay for hyaluronidase activity not requiring specialized reagents[END_REF]. In this assay biotinylated hyaluronan (substrate) is covalently coupled to Covalink-NH 96-well microtiter plates. After removal of the coupling solution and three washings with PBS supplemented with 2 M NaCl and 50 mM MgSO 4 , the plates were equilibrated with assay buffer (0.1 M formate, pH 3.7, 0.15 NaCl and 1% Triton X-100).

Conditioned medium from each sample supplemented with 1 µg/ml aprotinin was cleared by centrifugation, and dialyzed against 0.01 M formate, pH 3.7, 0.015 M NaCl containing aprotinin over-night at 4 o C. Cell layers extracted for 30 min with 1 ml of ice-cold 0.01 M formate, pH 3.7, 0.015 M NaCl and 0.1% Triton X-100 containing DNase I (0.5 mg/ml), aprotinin (1 µg/ml) and Pefabloc (0.5 mg/ml), were sheared through a 20 gauge needle and centrifuged at 10 000 x g for 15 min. Then, the samples were concentrated 10-fold and the protein content was measured by the BCA kit. Samples of conditioned media and of cell layers as well as Streptomyces hyaluronidase standards (1.0 to 1 x 10 -6 U/ml; Sigma-Aldrich) were diluted with the assay buffer at appropriate dilutions, and added in the Covalink plates with immobilized biotinylated hyaluronan. After 1 h of incubation, under shaking at 37 o C, the reaction was stopped with 6 M Guanidinium-HCl followed by three washes with PBS supplemented with 2 M NaCl, 50 mM MgSO 4 and 0.05% Tween. Then, the residual coupled biotinylated-hyaluronan was detected with streptavidin biotinylated HRP reaction and the complexes were visualized as described above.

Immunoblotting

Human dermal fibroblasts (4 x 10 5 cells in 10 cm dish) were cultured in DMEM containing 10% FBS for 24 hours. After incubation in starvation medium for 24 h, quiescent cells or cells stimulated with various concentrations of PDGF-BB (5 -50 ng/ml) or TGFß (2.5 and 5 ng/ml), were incubated in the absence or presence of 10 µM U0126 or 20 µM LY294002. At different time intervals after treatment, the medium was removed and cell layers were washed two times with ice-cold PBS followed by lysis in 50 µl of ice-cold RIPA buffer (50 mM Hepes, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1% NP-40, 0.1% SDS, 1% deoxycholate, 1 mM dithiothreitol) containing phosphatase and protease inhibitors (50 mM NaF, 1 mM Na 3 VO 4 , 0.5 mg/ml pefabloc, 10 µM leupeptin, 2 mM pepstatin and 1 µg/ml aprotinin) and mixing at 4 o C for 30 min. Cell lysates were scraped into microfuge tubes on ice, sheared by a 20 gauge needle, incubated an additional 30 min on ice and centrifuged at 14 000 x g for 15 min, at 4˚C, in order to remove insoluble debris. The protein content in the supernatant was measured by BCA kit (Pierce). Then, samples (40 µg protein per well) were mixed with one volume of reducing sample buffer, denatured at 95˚C for 3 min, cooled down for 15 min before addition of 20 mM iodoacetamide and separation on SDS-PAGE using 10% polyacrylamide gels, followed by transfer to nitrocellulose membranes (Amersham Biosciences, Uppsala, Sweden). Non-specific binding sites on the membrane were blocked

Real-time PCR

Total RNAs were obtained from human dermal fibroblast cultures (4 x 10 5 cells per 10 cm dish) incubated in starvation medium alone or in medium supplemented with PDGF-BB (50 ng/ml), U0126 (10 µM), LY294002 (20 µM), or combinations thereof, as well as 10% FBS. After 1, 4 and 24 h of treatment, the cells were washed twice with PBS and the total 1). Each reaction mixture, in addition to the specific primer, contained 1 µl of cDNA from RT reactions, 3.5 mM MgCl 2 , 200 µM dNTPs, SYBR ® Green I (1/66 000) and Hot Goldstar enzyme (0.025 units /µl); it was amplified for 40 cycles at an annealing temperature of 60°C using an ABI PRISM 7000

Sequence Detection System (SDS Software; Applied Biosystems). For each optimized assay, a relative standard curve was run which resulted in a linear plot of threshold values (C T , which reflects the cycle number at which statistically significant amplicon numbers have been accumulated above the baseline) toward log input total RNA (0 -100 ng); the slope values of each constructed standard curve were approximately equal (within 10% from the theoretical value of the slope which should not be lower than -3.3 [START_REF] Lekanne Deprez | Sensitivity and accuracy of quantitative real-time polymerase chain reaction using SYBR green I depends on cDNA synthesis conditions[END_REF]), indicating a similar amplification efficiency of each target. A dissociation curve analysis was performed to verify the presence of correct product at predicted melting temperatures and absence of primer dimers. Using Excel, the relative expression level of each target normalized to endogenous reference gene (GAPDH) and relative to a calibrator (sample exhibiting the lowest expression level, 1 x sample) was measured by using the comparative C T method (Applied Biosystems, User Bulletin #2).

Proliferation assay

Human dermal fibroblasts (1 x 10 4 cells per well in 24-well plates) were cultured for 24 h in complete medium followed by 48 h in starvation medium. At this time, fresh starvation medium was added in the absence or presence of PDGF-BB (3 -10 ng/ml), hyaluronan (5 or 25 µg/ml), and monoclonal antibodies against the hyaluronan receptor CD44 (100 µl Hermes-1 serum-free myeloma culture medium / ml which specifically inhibits the binding of Media were then aspirated, cells washed with PBS and incubated with 5% trichloroacetic acid for 20 min at 4 o C. Cell layers were washed twice with water followed by lysis in 400 µl of 1 M NaOH for 30 min at room temperature and neutralization with 500 µl of 1 M HCl. The precipitated DNA was then processed for scintillation counting.

RESULTS

PDGF-BB is the predominant stimulator of hyaluronan production in human dermal fibroblast cultures

Our previous studies revealed that PDGF-BB is a potent stimulator of hyaluronan synthesis in cells of mesenchymal origin, such as fibroblasts and mesothelial cells [START_REF] Heldin | Effect of growth factors on hyaluronan synthesis in cultured human fibroblasts[END_REF]33]. However, in order to investigate whether there are differencies in the hyaluronan synthesizing capacity of various fibroblast-derived cell lines, including primary fibroblast cultures derived from normal human dermis and from keloid-like dermal scars [34], as well as in a panel of epithelial cells, the PDGF-BB-mediated hyaluronan production by various connective tissue cell types was studied. The analysis revealed that untreated and PDGF-BB-treated 3T3 mouse embryo fibroblasts and human adult lung fibroblasts synthesized about 650 ng and 750 ng hyaluronan per 1 x 10 5 cells and 24 h, respectively, independently of PDGF-BB stimulation.

Notably, MEFs, 293 cells and Cos-1 cells synthesized only minute amounts of hyaluronan (Fig. 1). The hyaluronan content in 24 h-conditioned media from subconfluent cultures of human foreskin fibroblasts AG 1518 and AG1523, as well as fibroblasts derived from normal dermis or dermal keloid scars, were increased 2 -4-fold, when cells were stimulated with PDGF-BB (Fig. 1). Among the cells with an epithelial phenotype, primary human epidermal keratinocytes synthesized hyaluronan in a PDGF-BB-independent manner, consistent with the lack of PDGF receptors on these cells, whereas the synthesis of hyaluronan by the human epithelial cell line HaCat was stimulated by PDGF-BB. Notably, the mammary NMuMG cells did not synthesize any significant amounts of hyaluronan (Fig. 1).

Thus, the hyaluronan synthesizing capacity of fibroblasts derived from various origins, differs upon PDGF-BB stimulation; the primary cultures of human dermal fibroblasts exhibited the highest hyaluronan production in response to PDGF-BB, compared to other cells tested, and were thus chosen for further studies.

We next investigated the effects of other growth factors of importance for the biology of dermis, on hyaluronan synthesis at concentrations found to give maximum effects (data not shown). PDGF-BB stimulated hyaluronan synthesis in human dermal fibroblasts even more efficiently than 10% FBS (Fig. 2A). Epidermal growth factor (EGF) slightly increased the hyaluronan content in the conditioned 24 h media, whereas basic fibroblast growth factor (bFGF) and transforming growth factor-ß1 (TGF-ß1) had no effect. Addition of EGF together with PDGF-BB did not increase hyaluronan production appreciably over that synthesized in response to PDGF-BB alone, whereas addition of bFGF gave a slightly decreased effect.

Interestingly, addition of TGF-ß to PDGF-BB resulted in an about 72% decrease in the amount of hyaluronan compared to stimulation with PDGF-BB alone (Fig. 2A). These data support the notion that PDGF-BB has a major role in hyaluronan biosynthesis in dermis. The stimulatory effects of PDGF-BB were higher in primary subconfluent cultures compared with that of confluent cultures of human dermal fibroblasts (Fig. 2B).

PDGF-BB and TGF-ß1 affect differentially the enzymes involved in synthesis and degradation of hyaluronan

As a first step to further elucidate which of the three HAS isoforms accounts for the increased levels of hyaluronan in the conditioned media of dermal fibroblast cultures, in response to PDGF-BB, we determined the mRNA levels of HAS genes in non-stimulated and PDGF-BB stimulated cells using real-time PCR. Furthermore, to investigate whether the hyaluronan degradative enzymes also contributed to the modulation of the levels of hyaluronan in the conditioned media, the levels of mRNAs for HYAL1 and HYAL2 were also determined. The analysis of non-stimulated and PDGF-BB-stimulated dermal fibroblasts, at 1 h, 4 h and 24 h, revealed that dermal fibroblasts expressed only traces of HAS1, HAS3 and HYAL1 transcripts.

PDGF-BB stimulation of the cells triggered an early and significant induction (after 1 h of stimulation) of mRNAs for HAS1 (10-fold), HAS3 (7-fold) and HYAL1 (4-fold) compared with the corresponding basal transcript levels (p < 0.05). Longer treatments of cell cultures with PDGF-BB, led to a decline in the expression levels of HAS1 and HAS3, whereas the HYAL1 transcript was high even after 4 h of stimulation, followed by a decline at 24 h.

Independently of the degree of stimulation, the mRNA levels of HAS1, HAS3 and HYAL1

were very low (between 0.01% -0.07%) compared to GAPDH. Importantly, dermal fibroblasts exhibited a high basal expression of HAS2 and HYAL2 mRNA levels, about 900fold and 40-fold higher compared to HYAL1 (1 x sample) (Fig. 3). The already high transcriptional activity of HAS2 gene in non-stimulated cells, was induced about 3-fold after 4 h of stimulation with PDGF-BB; after longer treatment, the HAS2 gene expression still possessed transcriptional activity significantly higher compared to control levels (Fig. 3).

Notably, the endogenous high transcriptional activity of HYAL2 did not significantly change from 1 h to 24 h in response to PDGF-BB. Comparing the mRNA levels of the HAS2 and HYAL2 isoforms with those of GAPDH, revealed that the HAS2 and HYAL2 transcript levels were about 36% and 0.6% in PDGF-BB-stimulated cells, respectively, of those of GAPDH.

Thus, it is likely that the induction of the HAS2 gene and, to a lesser extent, the HAS1 and HAS3 genes, rather than the suppression of HYAL1 and HYAL2 genes, accounts for the increased hyaluronan levels in response to PDGF-BB in dermal fibroblast cultures.

We next investigated, using immunoblotting, the expressions of HAS2, HYAL1 and HYAL2 protein in 6 h PDGF-BB-stimulated and quiescent fibroblast cultures (Fig. 4). Bands of approximately 65 kDa (consistent with the size of HAS2) and about 50 kDa (consistent with the size of HYAL2) were seen in lysates of dermal fibroblasts when blots were incubated with polyclonal HAS2 or HYAL2 antibodies, respectively, but not when the antiserum was pre-incubated with a 200-fold excess of the respective peptides used for immunization.

Furthermore, a 50 kDa component, consistent with the size of HYAL1 [START_REF] Frost | HYAL1LUCA-1, a candidate tumor suppressor gene on chromosome 3p21.3, is inactivated in head and neck squamous cell carcinomas by aberrant splicing of pre-mRNA[END_REF][START_REF] Stern | Stimulation of hyaluronan biosynthesis by platelet-derived growth factor-BB and transforming growth factor-b1 involves activation of protein kinase C[END_REF], was detected using the 17E9 monoclonal antibodies against HYAL1 [37]. Despite the low amount of HYAL1 mRNA (Fig. 3), an appreciable amount of HYAL1 protein was expressed. PDGF-BBstimulated cells expressed more HAS2 protein (Fig. 4A) and synthesized more hyaluronan (Fig. 4D) compared to unstimulated cells, whereas neither the HYAL1 nor the HYAL2 proteins changed significantly (Figs 4B andC). In addition, PDGF-BB-stimulated dermal fibroblasts exhibited similar activity of hyaluronidase(s) present in cell-layer extracts and conditioned media as the non-stimulated cells (Fig. 4E). Thus, PDGF-BB-mediated stimulation of hyaluronan synthesis is due to upregulation of HAS2 expression.

Because treatment of dermal fibroblasts with TGF-ß suppressed the stimulatory effect of PDGF-BB on hyaluronan production (Fig. 2A), we investigated the effects of TGF-ß on the expression of HAS2, HYAL1 and 2 as well as on hyaluronidase activity present in cell-layer extracts and conditioned media. As shown in Fig. 5, TGF-ß stimulation did not have any effect on HAS2 protein expression or hyaluronan amount in 24 h conditioned media, neither did it affect HYAL1 and 2 expressions. Interestingly, however, stimulation with TGF-ß increased, in a dose-dependent manner, the hyaluronidase activity about 3 -4-fold in conditioned media and about 2 -3-fold in cell layer extracts (Fig. 5E). This data suggests that whereas TGF-ß does not affect the amount of HYAL1 and 2, it is a powerful stimulator of hyaluronidase activity in dermal human fibroblasts.

PDGF-BB-induced stimulation of hyaluronan synthesis requires active Erk1/2 and PI3K signaling pathways

In order to explore which signaling pathways are important for stimulation of hyaluronan biosynthesis in response to PDGF-BB, we used specific inhibitors of downstream signaling molecules at non-cytotoxic concentrations, as determined by the exclusion of trypan blue (data not shown). The PDGF-BB-induced 2.5-fold increase in the hyaluronan synthesis was completely prevented when the dermal fibroblasts were cultured in the presence of the MEK1/2 inhibitor U0126 that inhibits MEK, an activator of the MAP kinase Erk1/2, or in the presence of the PI3-kinase inhibitor LY294002 (Fig. 6A). Notably, addition of the specific Src inhibitor SU6656 and p38 inhibitor SB203580, only slightly suppressed hyaluronan production (less than 20% inhibition), indicating that these signaling pathways does not have a major role in PDGF-BB-induced hyaluronan synthesis. Thus, the powerful stimulatory effect of PDGF-BB on hyaluronan biosynthesis is dependent on activation of PI3-kinase and Erk1/2 signaling pathways.

Further studies on the more downstream signaling molecules involved in PDGF-BBmediated hyaluronan synthesis revealed an important role of NFkB. This was demonstrated by using the SN50 peptide inhibitor which is cell-membrane permeable and contains the nuclear localization sequence of NFkB and thereby inhibits the translocation of its active complex into the nucleus. PDGF-BB stimulation of hyaluronan synthesis was decreased about 80% by the SN50 peptide. In contrast, the control SN50M peptide, which has mutations in the nuclear localization sequence of NFkB and thus does not prevent translocation of the NFkB active complex into the nucleus, did not significantly inhibit the hyaluronan produced by PDGF-BB stimulation. These observations support the notion that NFkB has a central role in the activation of hyaluronan synthesis.

Also at the transcriptional level, PDGF-BB-induced HAS2 gene expression was completely prevented if the fibroblasts were cultured in the presence of the MEK inhibitor U0126, or in the presence of the PI3-kinase inhibitor LY294002; the effects of the inhibitors on PDGF-BB-induction of HAS1, HAS3 and HYAL2 were less apparent (Fig. 7). Furthermore, the levels of mRNAs for HYAL1 were also suppressed when these inhibitors were added to the cultures. These studies indicate an involvement of the Erk MAP kinase and PI3-kinase signaling pathways in HAS2 and HYAL1 transcriptional activities. A four hours stimulation of the cells with 10% FBS led to increases in HAS1, HAS2 and HAS3 transcripts; the increase in HAS2 transcriptional activity was similar to that detected after PDGF-BB stimulation. However, in addition, 10% FBS caused powerful inductions of HYAL1 and HYAL2 transcripts (Fig. 7).

Inhibition of hyaluronan binding to CD44 inhibits PDGF-BB-mediated proliferation of human dermal fibroblasts

In order to investigate whether hyaluronan is of importance for the PDGF-BB-stimulated growth stimulation of dermal fibroblasts, we subjected the cells to a [ 3 H]thymidine incorporation assay. As shown in Fig. 8A, PDGF-BB induced [ 3 H]thymidine incorporation in a dose-dependent manner. When unstimulated or PDGF-BB-stimulated fibroblasts were cultured in the presence of 5 or 25 µg/ml of hyaluronan, an increase in the PDGF-BBmediated cell growth was observed (Fig. 8B). Interstingly, this growth-promoting effect of hyaluronan on PDGF-BB-stimulated cells was inhibited in the presence of Hermes-1 antibodies that specifically block the binding of hyaluronan to CD44, but not in the presence of Hermes-3 antibodies that does not effect the binding of hyaluronan to CD44. Thus, binding of hyaluronan to CD44 receptor is important for the proliferative effect of PDGF-BB in dermal fibroblasts in culture.

DISCUSSION

In the present study, we show that among various epithelial and fibroblast cells from different origins, the adult human dermal fibroblasts synthesize the highest amounts of hyaluronan. We used primary cultures of dermal fibroblasts for a thorough analysis of the effects of PDGF-BB and TGF-ß1 on the mRNA and protein levels of HAS and HYAL isoforms. PDGF-BB potently stimulated hyaluronan synthesis (Figures 1,2), HAS2 gene transcriptional activity (Figures 3 and7), and HAS2 protein induction (Fig. 4A).

Importantly, we found that inhibition of hyaluronan binding to its receptor CD44 prevents PDGF-BB-induced [ 3 H]thymidine incorporation, suggesting that induction of hyaluronan is an important part of the mitogenic PDGF-BB response. Interestingly, we recently found that higher concentrations of hyaluronan (100 µg/ml) inhibits PDGF ß-receptor activation in a tyrosine phosphatase-dependent mechanism [25]. Thus, hyaluronan appears to affect PDGF stimulation in a biphasic manner; low concentrations of hyaluronan enhances, whereas high concentrations inhibits, PDGF stimulation.

Previous studies revealed that PDGF-BB and TGF-ß induce hyaluronan synthesis in cultures of human foreskin fibroblasts in an additive manner [38]. In contrast, we report here that in human dermal fibroblast cultures TGF-ß suppresses the PDGF-BB-induced hyaluronan levels (Fig. 2A). Most likely, the stimulatory effect of TGF-ß1 on hyaluronidase activity in dermal fibroblasts (Fig. 5E) contributes to the lowering of the hyaluronan levels.

Interestingly, the increase in hyaluronidase activity occurred without an increase in the amount of HYAL protein. It is possible that TGF-ß induces proteolytic activation of HYAL proteins or induces the synthesis of an activation co-factor. Furthermore, TGF-ß1 stimulation of normal human mesothelial cell cultures led to suppression of HAS2 gene expression but induction of HAS1 transcript [33]. Thus, the differential effects of TGF-ß1 on hyaluronan production and degradation is cell-type specific and involves regulation of both hyaluronan synthesizing and degrading enzymes. These differences may have functional implications since hyaluronan fragments have different effects on cells compared to high molecular weight hyaluronan, and e.g. stimulates angiogenesis in a CD44-and chemokine CXCL1-dependent manner [START_REF] Takahashi | Hyaluronan fragments induce endothelial cell differentiation in a CD44-and CXCL1/GRO1-dependent manner[END_REF].

Following activation of the PDGF ß-receptor by PDGF-BB, several signaling pathways are activated. Two of the best defined pathways, supporting cell survival and proliferation, are the Erk MAP kinase and PI3 kinase pathways (Fig. 9). Activation of Erk MAP kinase leads to activation of the 90 kDa ribosomal S6 kinases (RSKs), that are responsible for the phosphorylation of several substrates, such as the transcription factor cAMP response element binding protein (CREB) and the transcriptional inhibitor IkB, that upon phosphorylation is targeted for ubiquitination and degradation leading to activation of NFkB and its translocation to the nucleus and binding to specific promoter elements [START_REF] Shimamura | Rsk1 mediates a MEK-MAP kinase cell survival signal[END_REF]. The PI3 kinase pathway is also important in the PDGF-mediated anti-apoptotic signaling through Akt and the IkB kinase (IKK) leading to active NFkB complex [START_REF] Romashkova | NF-kappaB is a target of AKT in antiapoptotic PDGF signalling[END_REF]. As shown in Figures 6 and7, inhibition of either of these pathways using specific inhibitors results in inhibition of both the HAS2 transcriptional activity and hyaluronan synthesis, to a similar level as for the non-stimulated cells. Notably, the proximal promoter region of HAS2 gene has putative binding sites for the CREB and NFkB transcription factors [42] (Fig. 9) which are downstream of the Erk MAP kinase and PI3K signaling pathways. Our finding that inhibition of either of the Erk MAP kinase or PI3-kinase pathways is sufficient to inhibit PDGF-BBinduced hyaluronan synthesis, suggests that these pathways are necessary for induction of hyaluronan synthesis in dermal fibroblasts. More recent studies on the HAS2 promoter have also identified a functional STAT-responsive element [START_REF] Saavalainen | The human hyaluronan synthase 2 gene is a primary retinoic Acid and epidermal growth factor responding gene[END_REF]; STAT isoforms are also effector molecules downstream of the PDGF ß-receptor.

The composition of the extracellular matrix strongly influences the phenotypes of cells [START_REF] Christofori | Changing neighbours, changing behaviour: cell adhesion molecule-mediated signalling during tumour progression[END_REF], and thus has important pathobiological implications. Hyaluronan and hyaluronan-CD44

  with 5% defatted milk in Tris-buffered saline (TBS-T; 50 mM Tris HCl, pH 7.4, 200 mM NaCl, supplemented with 0.1% Tween-20), at room temperature for 1 h, followed by two washes in TBS-T. Then, the membranes were incubated either with phospho-p44/42 Erk MAP kinase (Thr202/Tyr204) rabbit monoclonal antibodies (p-Erk1/2; dilution 1:1000; Cell Signaling Technology #4377), phospho-Akt (Ser473) rabbit antiserum (p-Akt; dilution 1:1000; Cell Signaling Technology #9271), rat monoclonal antibody 17E9 against Hyal1 (2 µg/ml; kindly provided by Dr G. Frost, Sidney Kimmel Cancer Center, San Diego, USA), polyclonal HYAL2 antiserum (1:300 dilution), or rabbit anti-HAS2 IgG (5 µg/ml; HiTrap protein G HP purified HAS2 antiserum) in TBS-T buffer containing 1% BSA overnight at 4˚C. The endogenous levels of Erk1/2 and Akt were detected by incubating the membranes with rabbit polyclonal anti-Erk1/2 antiserum (1:250 dilution; kindly provided by Charlotte Rorsman, Ludwig Institute for Cancer Research, Uppsala) and anti-Akt antiserum (1:1000 dilution; Cell-Signaling Technology #9272), respectively. After five washes with TBS-T, the membranes were incubated either with anti-rabbit horseradish peroxidase IgG (1:3000 diluted in 5% defatted milk in TBS-T) or with anti-rat horseradish peroxidase IgG (1:1000 diluted in 5% defatted milk in TBS-T). Immunoreactive bands were detected by enhanced chemiluminescence, stripped with Stripping buffer (Pierce, #21059) and re-blotted with antibodies, as specified.

15 FIGURE LEGENDS Fig. 1 .

 15LEGENDS1 FIGURE LEGENDS

Fig. 2 .

 2 Fig. 2. Stimulation of hyaluronan synthesis by growth factors in cultures of human dermal fibroblasts at different cell densites (A) Quiescent human dermal fibroblasts (3 x 10 4 cells per well in 12-well plates) were incubated in the absence (medium containing 0.1% FBS) or presence of 10% FBS, PDGF-BB (50 ng/ml), EGF (10ng/ml), bFGF (5 ng/ml), TGF-β1 (5 ng/ml), or combinations thereof. (B) Subconfluent (1 x 10 4 cells per well in 12-well plates) or confluent (4 x 10 4 cells per well in 12-well plates) dermal fibroblast cultures were incubated in starvation medium, or in medium supplemented with 10% FBS or 50 ng/ml of PDGF-BB. Open columns, subconfluent cultures; filled columns, confluent cultures. The hyaluronan amount in the cell culture media was determined after 24 h. Data shown is from a representative experiment out of two separate experiments performed with similar results. Columns depict the average of duplicate determinations ± variation. * significantly different (p < 0.05) from non-stimulated cells.

Fig. 3 .

 3 Fig. 3. Time course of HAS and HYAL mRNAs expression in response to PDGF-BB Quiescent human dermal fibroblasts (4 x 10 5 cells per 10 cm dish) were incubated in the absence (medium containing 0.1% FBS) or presence of PDGF-BB (50 ng/ml) for 1 h, 4 h and 24 h. The relative expression levels of HAS and HYAL mRNAs were determined by real-time RT-PCR, as described in Materials and Methods. Columns, means of triplicate determinations ± SD. Data shown is a representative experiment out of two separate experiments with similar results. Open columns, non-stimulated cells; gray columns, PDGF-BB-stimulated cells. * The transcriptional activity for each PDGF-BB-induced HAS or HYAL gene is significantly different (p < 0.05) from non-stimulated cells.

Fig. 4 .

 4 Fig. 4. Effects of PDGF-BB on HAS2, HYAL1 and 2 protein expression levels, and activitiesQuiescent dermal fibroblasts (4 x 10 5 cells per 10 cm plate) were incubated in the absence (medium containing 0.1% FBS) or presence of PDGF-BB (5 -10 ng/ml) for 6 h followed by lysis as described in Materials and Methods. Cell lysates (40 µg per well) were subjected to SDS-PAGE using 10% acrylamide gels followed by transfer to nitrocellulose membrane. The blots were incubated with polyclonal antibodies against HAS2 protein (5 µg/ml) (A), or against HYAL2 protein (1: 300 dilution) (B), in the absence or presence of a 200 molar excess of the corresponding blocking peptides, respectively. In panel (C) the membrane was blotted with the monoclonal antibody 17E9 against HYAL1(2 µg/ml). Panels (D) and (E) shows the quantifications of hyaluronan content in conditioned media, as well as Hyals activities in conditioned media (open columns) and cell layers (filled columns), respectively. The data shown are representative of two separate experiments with similar results (A, B, C), and the average of two separate experiments (D, E). Columns, means of duplicates ± variation.

Fig. 5 .

 5 Fig. 5. Effects of TGF-ß1 on HAS2, HYAL1 and 2 protein expression levels and activitiesLysates (40 µg per well) from quiescent or TGF-ß1-stimulated cells were subjected to SDS-PAGE using 10% acrylamide gels followed by transfer to nitrocellulose membrane. The blots were incubated with polyclonal antibodies against HAS2 protein (5 µg/ml) (A), against HYAL2 protein (1: 300 dilution) (B), in the absence or presence of a 200 molar excess of the corresponding blocking peptides, or against the monoclonal antibody 17E9 against HYAL1 (2 µg/ml) (C). (D) shows the quantifications of hyaluronan content in conditioned media, and (E) hyaluronidase activity in conditioned media (open columns) and cell layers (filled columns), respectively. The data shown are representatives of two separate experiments with similar results (A, B, C), and the average of two separate experiments (D, E). Bars, means of duplicates ± variation.

Fig. 6 .

 6 Fig. 6. Effects of inhibitors on hyaluronan synthesis by dermal fibroblastsQuiescent dermal fibroblasts (3 x 10 4 cells per well in 12-well plates) were cultured in the absence (medium containing 0.1% FBS) or presence of PDGF-BB (50 ng/ml), the Src inhibitor SU6656 (2 µM), the p38 inhibitor SB203580 (10 µM), the MEK1/2 inhibitor U0126 (10 µM), the PI3-K inhibitor LY294002 (20 µM), the NFκB inhibitor SN50 (16 µM) and its control inhibitory peptide SN50M (16 µM), and 10% FBS or combinations thereof for 24 h. Hyaluronan content in conditioned media was measured as described in Materials and

Fig. 7 .

 7 Fig. 7. HAS and HYAL mRNA expression in response to PDGF-BB and inhibitors Quiescent dermal fibroblasts (4 x 10 5 cells per 10 cm dish) were treated with PDGF-BB (50 ng/ml), MEK1/2 inhibitor U0126 (10 µM) and PI3 kinase inhibitor LY294002 (20 µM), alone or in combinations, for 4 h. The relative expressions levels of HAS and HYAL mRNAs were determined by real-time PCR, as described in Materials and Methods. Bars, means of triplicate determinations ± SD. Data shown is from a representative experiment of two separate experiments with similar results. * p < 0.01 compared to control.

Fig. 8 .

 8 Fig. 8. PDGF-BB-mediated incorporation of [ 3 H]thymidine to fibroblast cultures is promoted by hyaluronan binding to CD44Cells (1 x 10 4 /well in 12-well plates) after a 48 h of starvation, were cultured for an additional 16 h in starvation medium alone or in medium containing PDGF-BB (3-10 ng/ml), hyaluronan (5 or 25 µg/ml), Hermes-1 (100 µl/ml), Hermes-3 (50 µg/ml) or combinations thereof. Then, [ 3 H]thymidine was added and the cultures were incubated for 6 h at 37 o C, as described in Materials and Methods. In panel (A), PDGF-BB-induced [ 3 H]thymidine incorporation in a dose-depenent manner is depicted. The data is a representative experiment out of two performed. In panel (B), hyaluronan addition alone promoted PDGF-BB-induced [ 3 H]thymidine incorporation, and, its addition together with Hermes-1 that blocks the binding of hyaluronan to CD44 suppressed PDGF-BB growth promoting capacity. Open columns, unstimulated cells; filled columns, PDGF-BB-stimulated fibroblasts (3 ng/ml). The data is the average of two separate experiments. Bars both in (A) and (B) are means of triplicate determinations ± SD.

Fig. 9 .Fig. 1 BiochemicalBiochemical

 91 Fig. 9. Schematic illustration of signaling pathways downstream of the PDGF ß-receptor that are involved in HAS2 gene regulation

Biochemical Journal Immediate Publication. Published on 27 Feb 2007 as manuscript BJ20061757 © 2007 The Authors Journal compilation © 2007 Biochemical Society RNAs

  were extracted by using RNAqueous TM -4PCR Kit (Ambion #1914). Each of the total RNAs were further treated with DNase I, followed by DNase inactivation (Ambion #1914), in order to obtain DNA-free total RNA. The purity of the RNAs was verified by measurement of 260/280 nm OD values (ratio ≥2.0), and their integrity were determined by separation in a 1%

	agarose formaldehyde gel, followed by inspection under UV light. Then, each purified RNA
	(2 µg RNA in 20 µl of total reaction volume) was reverse-transcribed into the corresponding
	cDNA by SuperScript TM II RNase H -Reverse Transcriptase (Invitrogen #18064-014),
	followed by real-time PCR using qPCR TM Core kit for SYBR ® Green I (Eurogentec # RT-
	SN73-05), according to the instruction of the manufacturer. The primers for the target genes
	(HAS1, 2, 3 and HYAL1, 2; final concentration of 300 nM) and reference gene GAPDH (final
	concentration 100 nM) were designed by Primer Express ® Software v 2.0 (Applied
	Biosystems) and synthesized by Applied Biosystems (Table

Biochemical Journal Immediate Publication. Published on 27 Feb 2007 as manuscript BJ20061757 © 2007 The Authors Journal compilation © 2007 Biochemical Society hyaluronan

  to the CD44 or 50 µg Hermes-3 IgG / ml, ammonium sulfate precipitate; generously provided by professor Sirpa Jalkanen, Turku, Finland), or combinations thereof, and 16 h later the cells were treated with a 6 h pulse of 0.2 µCi of [ 3 H]thymidine per well.

Biochemical Journal Immediate Publication. Published on 27 Feb 2007 as manuscript BJ20061757 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 27 Feb 2007 as manuscript BJ20061757 © 2007 The Authors Journal compilation © 2007 Biochemical Society

Biochemical Journal Immediate Publication. Published on 27 Feb 2007 as manuscript BJ20061757 © 2007 The Authors Journal compilation © 2007 Biochemical Society

This work was supported by grants from the Swedish Cancer Society and the Faculty of Medicine at Uppsala University.