Dr Angel Nadal
email: nadal@umh.es

Paloma Alonso-Magdalena

Sergi Soriano

Ivan Quesada

Ana B Ropero

THE PANCREATIC -CELL AS A TARGET OF ESTROGENS AND XENOESTROGENS: IMPLICATIONS FOR BLOOD GLUCOSE HOMEOSTASIS AND DIABETES

Keywords: endocrine disruptors, pregnandcy, β-cell failure, pollutants, environmental estrogens

The estrogen receptor ER is emerging as a key molecule involved in glucose and lipid metabolism. The main functions of pancreatic -cells are the biosynthesis and release of insulin, the only hormone that can directly decrease blood glucose levels.

Estrogen receptors ER and ER exist in

The role of the endocrine pancreas in blood glucose homeostasis

Blood glucose levels must be maintained within a precise range throughout the day, independently of dietary nutrient ingestion. This process, defined as blood glucose homeostasis involves a complex communication between different tissues, including the liver, skeletal muscle, adipose tissue, brain and the endocrine pancreas [START_REF] Herman | Glucose transport and sensing in the maintenance of glucose homeostasis and metabolic harmony[END_REF][START_REF] Rosen | Adipocytes as regulators of energy balance and glucose homeostasis[END_REF][START_REF] Fritsche | How insulin receptor substrate proteins regulate the metabolic capacity of the liver--implications for health and disease[END_REF]. During the fasting state, insulin remains at low levels because plasma glucose concentration is low. In this situation, the levels of the counter-regulatory hormones, glucagon, adrenaline and corticosteroids are increased to promote hepatic glucose production. In contrast, insulin, the only hormone in the body able to decrease blood glucose levels, is increased during the fed state. Insulin decreases blood glucose by promoting adipocyte and muscle glucose uptake, as well as by preventing the liver from producing glucose by suppressing glycogenolysis and gluconeogenesis [START_REF] Fritsche | How insulin receptor substrate proteins regulate the metabolic capacity of the liver--implications for health and disease[END_REF].

The endocrine cells of the pancreas are grouped in the islets of Langerhans, which constitute approximately 1 to 2% of the pancreas mass. Islets are distributed throughout the organ and in healthy human adults; their number reaches one million islets per pancreas. Each islet contains between 1000-3000 cells of five types, the most abundant are -cells, which synthesize and release insulin and -cells which synthesize and secrete glucagon. In a lower proportion, islets contain -cells and PP-cells that release somatostain and pancreatic polypeptide respectively [START_REF] Brissova | Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy[END_REF][START_REF] Cabrera | The unique cytoarchitecture of human pancreatic islets has implications for islet cell function[END_REF]. Recently, a small population of ghrelin-producing cells, named as epsiloncells, has been identified [START_REF] Prado | Ghrelin cells replace insulin-producing beta cells in two mouse models of pancreas development[END_REF]. The percentage of the different cell subpopulations and the islet cytoarchitecture vary between species. In rodent islets, cells are the most abundant, 60-80 % of the total number of cells; -cells account for 15-20 %, while -cells are less than 10% and PP-cells about 1%. In rodents, -cells are in the core of the islets while non--cells are distributed in the periphery [START_REF] Brissova | Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy[END_REF][START_REF] Cabrera | The unique cytoarchitecture of human pancreatic islets has implications for islet cell function[END_REF]. In human islets, the proportion of and PP-cells is similar to rodent islets, but the percentage of -cells is higher (35-45%) and the -cell percentage is lower (55-65%). Moreover,  and -cells are distributed evenly throughout the islet, suggesting that paracrine interactions between both types of cells may be more important in humans that in rodents [START_REF] Brissova | Assessment of human pancreatic islet architecture and composition by laser scanning confocal microscopy[END_REF][START_REF] Cabrera | The unique cytoarchitecture of human pancreatic islets has implications for islet cell function[END_REF].

The main function of pancreatic -cells is the biosynthesis and release of insulin. The rate of insulin biosynthesis is controlled by many signalling molecules including: neurotransmitters, hormones and nutrients, among which glucose is the most important.

Normally, in response to short glucose stimulation, insulin biosynthesis is regulated by the increased translation of the preproinsulin transcript. After a prolonged glucose exposure, however, it is regulated via the insulin gene transcription [START_REF] Orland | Modulation of proinsulin messenger RNA after partial pancreatectomy in rats. Relationships to glucose homeostasis[END_REF][START_REF] Permutt | Analysis of the insulin gene in noninsulindependent diabetes[END_REF][START_REF] Poitout | Regulation of the insulin gene by glucose and fatty acids[END_REF]. Both transcriptional and translational regulation of insulin biosynthesis is essential to maintain the intracellular stores of insulin on a long term basis.

The secretory response of -cells depends on their electrical activity. This consists of oscillations of the membrane potential that range from electrically silent periods to depolarised plateaus on which Ca 2+ -action potentials originate [START_REF] Rorsman | The Cell Physiology of Biphasic Insulin Secretion[END_REF].

The classical stimulus-secretion coupling that drives insulin release involves the closure of K ATP channels by increasing the intracellular ATP/ADP ratio [START_REF] Ashcroft | Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells[END_REF] and diadenosine polyphosphates concentration (DPs) [START_REF] Ripoll | Diadenosine polyphosphates. A novel class of glucose-induced intracellular messengers in the pancreatic beta-cell[END_REF] because of glucose metabolism. The K ATP channels closure induces membrane depolarization that activates voltage-operated Ca 2+ channels and Ca 2+ influx [START_REF] Valdeolmillos | The relationship between glucose-induced K+ATP channel closure and the rise in [Ca2+]i in single mouse pancreatic beta-cells[END_REF].

Therefore, as a consequence of the oscillations in the membrane potential, a [Ca 2+ ] i oscillatory pattern is originated [START_REF] Nadal | Homologous and heterologous asynchronicity between identified alpha-, beta-and delta-cells within intact islets of Langerhans in the mouse[END_REF][START_REF] Santos | Widespread synchronous [Ca2+]i oscillations due to bursting electrical activity in single pancreatic islets[END_REF][START_REF] Valdeolmillos | Glucose-induced oscillations of intracellular Ca2+ concentration resembling bursting electrical activity in single mouse islets of Langerhans[END_REF], which triggers a pulsatile insulin secretion [START_REF] Barbosa | Control of pulsatile 5-HT/insulin secretion from single mouse pancreatic islets by intracellular calcium dynamics[END_REF][START_REF] Gilon | Oscillations of secretion driven by oscillations of cytoplasmic Ca2+ as evidences in single pancreatic islets[END_REF][START_REF] Dyachok | Glucose-induced cyclic AMP oscillations regulate pulsatile insulin secretion[END_REF].

Estrogens, estrogen receptors and blood glucose homeostasis

The evolution of type 2 diabetes requires the presence of defects in both insulin secretion (-cell dysfunction) and insulin action (insulin resistance). Both defects have a recognized genetic background as well as an environmental component, where the lack of exercise and obesity play important roles. Although it is not usually emphasized, during the prediabetic phase, when insulin resistance has developed, the -cell hyper secretes insulin to maintain normal blood glucose levels provoking hyperinsulinaemia [START_REF] Mcgarry | Banting lecture 2001: dysregulation of fatty acid metabolism in the etiology of type 2 diabetes[END_REF]. This hyperinsulinemia may produce an excess of insulin signaling in the liver, kidneys and ovaries, leading to hypertriglyceridemia, increased sodium retention, hypertension, and hyperandrogenism [START_REF] Biddinger | From mice to men: insights into the insulin resistance syndromes[END_REF].

Estrogens are hormones to be considered in blood glucose homeostasis, although in which conditions they exert a beneficial or detrimental action is still a matter of debate.

A change in blood estrogens occurs during pregnancy as well as during the menstrual cycle in humans or the estrous cycle in rodents. At those physiological levels, E2 is thought to be involved in maintaining normal insulin sensitivity and to be beneficial for β-cell function [START_REF] Livingstone | Sex steroids and insulin resistance[END_REF][START_REF] Louet | Antidiabetic actions of estrogen: insight from human and genetic mouse models[END_REF]. However, E2 levels above or below the physiological range may promote insulin resistance and type II diabetes [START_REF] Ding | Plasma sex steroid hormones and risk of developing type 2 diabetes in women: a prospective study[END_REF][START_REF] Godsland | Oestrogens and insulin secretion[END_REF][START_REF] Gonzalez | Dose-and time-dependent effects of 17beta-oestradiol on insulin sensitivity in insulin-dependent tissues of rat: implications of IRS-1[END_REF][START_REF] Livingstone | Sex steroids and insulin resistance[END_REF].

Evidence that high E2 concentrations are detrimental for blood glucose homeostasis has existed for a long time. In humans, the most consistent effects of oral contraceptives and estrogen replacement therapy (HRT) are lowering fasting plasma glucose and worsening glucose tolerance. However, basal insulin levels are unchanged or slightly reduced, whereas glucose-stimulated insulin release is enhanced [START_REF] Godsland | Oestrogens and insulin secretion[END_REF]. This is not only seen in humans but also in male mice exposed to E2 (100 µg/kg/day) for 4 days [START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF]Ropero et al., 2008a). Therefore, an excess of estrogens, such as in oral contraceptives or HRT with high estrogen doses, is related to insulin resistance [START_REF] Godsland | Oestrogens and insulin secretion[END_REF].

Low levels of estrogen because of ovariectomy or menopause are associated with impaired glucose tolerance and insulin resistance. This effects are counteracted in both situations by physiological estrogen replacement [START_REF] Godsland | The influence of female sex steroids on glucose metabolism and insulin action[END_REF]. It has been reported that estrogen function deficiency in men, due to the absence of ER or aromatase, results in impaired glucose metabolism. All patients with aromatase deficiency, due to a point mutation of the aromatase gene, have glucose metabolism impairment and insulin resistance [START_REF] Zirilli | Human models of aromatase deficiency[END_REF]. Similar abnormalities were found in aromatase-deficient (ArKO) mice [START_REF] Jones | Aromatase-deficient (ArKO) mice have a phenotype of increased adiposity[END_REF]. In the only described case of a congenital estrogen deficiency due to an inactivating mutation of the ER gene in a human, the man had glucose intolerance and hyperinsulinaemia, among other pathologies. In addition, he had a clear hormonal resistance syndrome, consistent of high serum E2, estrone, FSH and LH levels, although testosterone levels were normal [START_REF] Smith | Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man[END_REF][START_REF] Zirilli | Human models of aromatase deficiency[END_REF]. In humans it has been shown that genetic polymorphism of ER gene is associated with type II diabetes and metabolic syndrome [START_REF] Gallagher | Association of the estrogen receptor-alpha gene with the metabolic syndrome and its component traits in African-American families: the Insulin Resistance Atherosclerosis Family Study[END_REF]. It has been demonstrated that estrogen receptors ER and ER have both been involved in energy balance and blood glucose homeostasis (Ropero et al., 2008b). Nevertheless, evidence points to ER as the main mediator. ER knockout mice (ERKO) are obese and insulin resistant [START_REF] Heine | Increased adipose tissue in male and female estrogen receptor-alpha knockout mice[END_REF]. ER has been involved in glucose metabolism in different tissues including skeletal muscle, adipose tissue, liver, brain and endocrine pancreas [START_REF] Barros | Estrogen receptors: new players in diabetes mellitus[END_REF]Ropero et al., 2008b).

GLUT4 is the major insulin-stimulated glucose transporter and the main rate-limiting step in insulin-stimulated glucose uptake in muscle and adipocytes. GLUT4 expression in the skeletal muscle cell membrane depends on ER. It is extremely reduced in ERKO mice, unaffected in ERKO mice and elevated in ArKO mice [START_REF] Barros | Estrogen receptors: new players in diabetes mellitus[END_REF].

The estrogen receptor ER is also involved in modulating hepatic insulin sensitivity, but the ER is not. Actually, ERKO mice have severe hepatic insulin resistance as well as a concomitant alteration of glucose uptake by skeletal muscle [START_REF] Bryzgalova | Evidence that oestrogen receptor-alpha plays an important role in the regulation of glucose homeostasis in mice: insulin sensitivity in the liver[END_REF]. Additionally, estrogens control the distribution of body fat and adipose tissue metabolism, involving both ER and ER [START_REF] Cooke | The role of estrogen and estrogen receptor-alpha in male adipose tissue[END_REF][START_REF] Couse | Estrogen receptor null mice: what have we learned and where will they lead us?[END_REF][START_REF] Naaz | Effect of ovariectomy on adipose tissue of mice in the absence of estrogen receptor alpha (ERalpha): a potential role for estrogen receptor beta (ERbeta)[END_REF][START_REF] Penza | Genistein affects adipose tissue deposition in a dose-dependent and gender-specific manner[END_REF]. Indeed, it is known that estrogens can regulate the amount of white adipose tissue (WAT) both in females and males. It has been demonstrated that the absence of ERα produces adipocyte hyperplasia and hypertrophy in WAT, but not in brown adipocyte tissue (BAT). This is accompanied by insulin resistance and glucose intolerance [START_REF] Cooke | The role of estrogen and estrogen receptor-alpha in male adipose tissue[END_REF][START_REF] Heine | Increased adipose tissue in male and female estrogen receptor-alpha knockout mice[END_REF].

Human studies also suggest that both receptors may play an important role in fat metabolism and obesity. The ratio ER/ER seems to be associated with obesity as well as with the serum level and the production of leptin in omental adipose tissue in women [START_REF] Shin | The ratio of estrogen receptor alpha to estrogen receptor beta in adipose tissue is associated with leptin production and obesity[END_REF]. In the brain, the disruption of the ER in the ventromedial nucleus of the hypothalamus leads to weight gain, increased visceral adiposity, hyperphagia, hyperglycaemia and impaired energy expenditure in female mice [START_REF] Musatov | Silencing of estrogen receptor alpha in the ventromedial nucleus of hypothalamus leads to metabolic syndrome[END_REF].

In addition, ER has been shown to have anorectic effects mediated via the central nervous system [START_REF] Liang | Estrogen receptor beta is involved in the anorectic action of estrogen[END_REF]. Recently, it has been demonstrated that ER is important for adiposity regulation when mice are challenged with a high fat diet [START_REF] Foryst-Ludwig | Metabolic actions of estrogen receptor beta (ERbeta) are mediated by a negative cross-talk with PPARgamma[END_REF].

Although ER and ER represent the main link between estrogens and metabolism, other feasible players currently emerging are liver X receptors (LXRs). It is already known that estrogens can regulate LXRs in metabolic tissues. Lundholm et al 2004, demonstrated that 17 -estradiol can decrease mRNA expression of LXR. Furthermore they show a direct down-regulation of the LXR promoter by estrogen.

LXRs are well recognized as important regulators of cholesterol homeostasis as well as modulators of lipid and carbohydrate metabolism. Accordingly, it has been shown that the administration of synthetic LXR agonists to a mouse model of obesity and insulin resistance improves glucose tolerance [START_REF] Laffitte | Activation of liver X receptor improves glucose tolerance through coordinate regulation of glucose metabolism in liver and adipose tissue[END_REF]. This was attributed to a coordinate transcriptional regulation of genes involved in glucose metabolism in both the liver and the adipose tissue. In the liver, the activation of LXRs leads to the suppression of the gluconeogenic program. However, in white adipose tissue, the expression of the insulin-sensitive glucose transporter (GLUT4), which promotes glucose uptake, is upregulated after LXR activation [START_REF] Dalen | Expression of the insulin-responsive glucose transporter GLUT4 in adipocytes is dependent on liver X receptor alpha[END_REF].

Besides its action on peripheral tissues, a novel role for LXR in the pancreas has recently been described. The addition of the LXR agonist T09011317 to pancreatic islets results in enhanced glucose-dependent insulin secretion [START_REF] Efanov | Liver X receptor activation stimulates insulin secretion via modulation of glucose and lipid metabolism in pancreatic beta-cells[END_REF]. On the other hand, the absence of LXR in mice leads to glucose intolerance and to an accumulation of lipid droplets in pancreatic islets, attributed to a possible disturbance in cholesterol efflux due to a decreased expression of ABC transporters [START_REF] Gerin | LXRbeta is required for adipocyte growth, glucose homeostasis, and beta cell function[END_REF].

The role of the estrogen receptor ER in the pancreatic -cell

As described in the above paragraph, the role of estrogen receptors in glucose metabolism has been mainly studied in tissues other than the endocrine pancreas. In order to understand how estrogens affect the adaptation of -cells to physiological situations such as pregnancy and how they are involved in gender functional differences in the endocrine pancreas [START_REF] Karlsson | Gender difference in the glucagon response to glucopenic stress in mice[END_REF], it is essential to investigate the role of ER and ER in this cellular system.

In vivo experiments demonstrated that adult male mice treated with E2 100 µg/kg/day for 4 days presented an altered glucose tolerance and were insulin resistant. In addition, these animals presented a postprandial hyperinsulinaemia [START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF]Ropero et al., 2008b). When islets from treated animals were analyzed, they presented higher insulin content together with an enhanced insulin release in response to increasing glucose concentrations [START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF]. These may result from an adaptation of islets to the insulin resistance noted in these animals or to a direct effect of E2 on -cell insulin content or both.

In order to investigate whether estrogens directly regulate insulin biosynthesis, we studied pancreatic insulin content and insulin secretion in isolated islets from male mice cultured in the presence of estrogens. We obtained that physiological concentrations of E2 (100pM-10nM) together with a stimulatory glucose concentration 11mM, increased pancreatic insulin content in an inverted-U dose dependent manner [START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF]. This increase in pancreatic insulin content was not due to an enhanced cell mass, since the islet area did not change and no BrdU incorporation occurred during the estrogen treatment. Moreover, real time PCR experiments demonstrated that E2 treatment increased insulin mRNA. Therefore, E2 increases insulin gene expression and insulin biosynthesis, incrementing pancreatic insulin content. In addition, insulin secretion in response to glucose was enhanced as previously described [START_REF] Adachi | Promoting insulin secretion in pancreatic islets by means of bisphenol A and nonylphenol via intracellular estrogen receptors[END_REF].

In both in vitro and in vivo studies, E2 actions were blocked by the antiestrogen ICI182,780 suggesting that either ER or ER are involved [START_REF] Adachi | Promoting insulin secretion in pancreatic islets by means of bisphenol A and nonylphenol via intracellular estrogen receptors[END_REF][START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF][START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF]. The existence of estrogen receptors in -cells has not been extensively studied until now. Nonetheless, it has been demonstrated that both ER and ER were expressed in mouse -cells in primary culture by immunocytochemistry using a battery of antibodies against both receptors [START_REF] Nadal | Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to estrogen receptor alpha and estrogen receptor beta[END_REF]. In mice expressing human amyloid peptide, only a 58kDa form of ER was detected, but ER was not [START_REF] Geisler | Estrogen can prevent or reverse obesity and diabetes in mice expressing human islet amyloid polypeptide[END_REF]. In a recent study, the full-length 67kDa isoform was expressed in mouse and MIN6 cells, while a smaller 58kDa form was only present in islets (Le [START_REF] May | Estrogens protect pancreatic beta-cells from apoptosis and prevent insulin-deficient diabetes mellitus in mice[END_REF]. We demonstrated by RT-PCR and inmunocytochemistry that both ER and ER were present in mouse islets of Langerhans [START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF]. The use of ER and ER agonists as well as islets from ERKO and ERKO mice demonstrated that E2 action on insulin biosynthesis is mediated by the estrogen receptor ER [START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF].

Remarkably, the E2-induced increase of insulin biosynthesis was imitated by bisphenol A (BPA) at exactly the same doses of E2 and following the same pathway.

Besides the role that ER plays in the regulation of pancreatic insulin content, it has been demonstrated that it plays a key function in pancreatic -cell survival after an oxidative stress treatment (Le [START_REF] May | Estrogens protect pancreatic beta-cells from apoptosis and prevent insulin-deficient diabetes mellitus in mice[END_REF]. In this work, a single dose of streptozotocin (STZ) induced a loss of -cells and pancreas insulin content in wild type males as well as females. In ArKO-/-mice the same STZ treatment provoked -cell destruction in both genders, an effect that was suppressed by E2 treatment. These authors demonstrated that the protective effect of E2 is via ER. In the rat, however, diabetes development in response to STZ treatment is more severe in females than in males [START_REF] Vital | Sexual dimorphism in insulin sensitivity and susceptibility to develop diabetes in rats[END_REF]. After 4 weeks of diabetes, females show less and smaller pancreatic islets, and less serum insulin than control females and diabetic males; indeed diabetic females have higher blood glucose values and lower survival rates than diabetic males [START_REF] Vital | Sexual dimorphism in insulin sensitivity and susceptibility to develop diabetes in rats[END_REF]. This is in accordance with the action of testosterone in protecting -cells from STZ-induced apoptosis [START_REF] Morimoto | Protective effect of testosterone on early apoptotic damage induced by streptozotocin in rat pancreas[END_REF]. More research is warranted in this area to solve the gender differences that apparently exist between mice and rats.

E2 and BPA are equally effective through a non-classical estrogen-activated pathway

BPA is an organic compound widely recognised as an important endocrine disruptor chemical (EDC). This term refers to all those chemicals able to mimic or interfere with the normal actions of endocrine hormones. Although research was first focused only on reproductive parameters and potential carcinogenic effects, nowadays, many studies point out that EDC may affect multiple organ systems including the cardiovascular and neuro-endocrine systems. Moreover, recent interest has emerged from the idea of an association between EDCs and the metabolic syndrome. Regarding this issue, an increasing number of studies reports that exposure to chemicals during critical periods of differentiation, at low environmentally-relevant doses, alters developmental programming, resulting in obesity [START_REF] Newbold | Effects of endocrine disruptors on obesity[END_REF]. Furthermore, some persistent organic pollutants (POPs) have been reported to be linked to the prevalence of type 2 diabetes and insulin resistance [START_REF] Lee | Relationship between serum concentrations of persistent organic pollutants and the prevalence of metabolic syndrome among non-diabetic adults: results from the National Health and Nutrition Examination Survey 1999-2002[END_REF]. Remarkably, there exists an epidemiological link between BPA concentration in urine and type 2 diabetes, cardiovascular disease and liver enzyme abnormalities in a representative sample of adult US population [START_REF] Lang | Association of urinary bisphenol A concentration with medical disorders and laboratory abnormalities in adults[END_REF][START_REF] Vom Saal | Bisphenol A and risk of metabolic disorders[END_REF].

We have contributed to this hypothesis by analyzing the effects of EDCs on an essential tissue in glucose homeostasis, the endocrine pancreas, and reported the high sensitivity of this organ to BPA. Indeed, we have observed that BPA can mimic E2 effects with the same potency [START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF][START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF]Ropero et al., 2008b).

The equal potency of both E2 and BPA in insulin biosynthesis regulation was a paradox. In the past, BPA was considered a "weak" estrogen because of its low binding affinity to both ERα and ER [START_REF] Kuiper | Comparison of the ligand binding specificity and transcript tissue distribution of estrogen receptors alpha and beta[END_REF][START_REF] Kuiper | Interaction of estrogenic chemicals and phytoestrogens with estrogen receptor beta[END_REF], as well as for having a low transcriptional activity through these ERE binding receptors [START_REF] Sheeler | Environmental estrogens induce transcriptionally active estrogen receptor dimers in yeast: activity potentiated by the coactivator RIP140[END_REF]. In our work, however, both E2 and BPA were equally effective on insulin content up-regulation even though that the mouse insulin gene does not contain an ERE.

Examples of low dose effects of BPA have been reported in a wide variety of tissues and cell types [START_REF] Vom Saal | An extensive new literature concerning low-dose effects of bisphenol A shows the need for a new risk assessment[END_REF][START_REF] Wetherill | In vitro molecular mechanisms of bisphenol A action[END_REF], although the mechanism of action to explain them are still unclear. One explanation could be that BPA may act via ER through mechanisms other than binding to ERE. For instance, it may act through other transcription factors binding to their respective response elements [START_REF] Dahlman-Wright | International Union of Pharmacology. LXIV. Estrogen receptors[END_REF]. In addition, it has been demonstrated that extra-nuclear ER can activate protein kinases [START_REF] Giretti | Extranuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion[END_REF][START_REF] Migliaccio | Tyrosine kinase/p21ras/MAP-kinase pathway activation by estradiol-receptor complex in MCF-7 cells[END_REF][START_REF] Simoncini | Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase[END_REF]. Contrary to the results shown by Le [START_REF] May | Estrogens protect pancreatic beta-cells from apoptosis and prevent insulin-deficient diabetes mellitus in mice[END_REF] that exclusively found ER in -cell nuclei, our data indicate that in -cells, ER is located inside and outside the nucleus [START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF]. Furthermore, we found that the ER agonist PPT rapidly increased the phosphorylation of ERK1/2 in pancreatic -cell cytoplasm, with a maximal effect after 15 minutes of incubation (Alonso-Magdalena et al., 2008). The inhibition of ERK1/2 phosphorylation by PD98059 abrogated the action of PPT, E2 and BPA on islet insulin content [START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF]. The kinase Src may play a role as well, since the Src inhibitor PP1 greatly diminished the stimulatory effect of BPA on islet insulin content. Our results demonstrate that ERK1/2 mediate the ER-induced amplification of insulin biosynthesis (Figure 1). Additionally, estrogens and xenoestrogens act through other mechanisms independently of ER and ER to increase insulin secretion in a rapid manner in vitro and in vivo [START_REF] Nadal | Nongenomic actions of estrogens and xenoestrogens by binding at a plasma membrane receptor unrelated to estrogen receptor alpha and estrogen receptor beta[END_REF][START_REF] Nadal | Rapid insulinotropic effect of 17beta-estradiol via a plasma membrane receptor[END_REF][START_REF] Nadal | Estrogen and xenoestrogen actions on endocrine pancreas: from ion channel modulation to activation of nuclear function[END_REF][START_REF] Ripoll | Rapid regulation of pancreatic alpha-and beta-cell signalling systems by estrogens[END_REF][START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF].

Physiological and pathophysiological consequences of ER up-regulation of insulin

The action of ER on insulin biosynthesis has important physiological implications.

During pregnancy, islets adapt to the increased demand for insulin undergoing major changes in their structure and function. Among these changes, the increase of insulin synthesis is of great importance [START_REF] Bone | Mitabolic adaptation to pregnancy shown by increased biosynthesis of insulin in islets of Langerhans isolated from pregnant rat[END_REF][START_REF] Green | Regulation of insulin release from isolated islets of Langerhans of the rat in pregnancy. The role of adenosine 3':5'-cyclic monophosphate[END_REF]. Up until now, this effect has been attributed to the activity of lactogenic hormones [START_REF] Parsons | Adaptation of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion[END_REF][START_REF] Sorenson | Effects of steroid and lactogenic hormones on islets of Langerhans: a new hypothesis for the role of pregnancy steroids in the adaptation of islets to pregnancy[END_REF]. However, the E2-induced up-regulation of insulin biosynthesis may be another mechanism that, together with placental lactogen and prolactin, participates in the adaptation of insulin production to counteract the increased metabolic demand of pregnancy. It is plausible that, during pregnancy, ER integrates information from E2, glucose and other nutrients in the blood to regulate insulin gene expression and, therefore, contributes to the maintenance of insulin and glucose homeostasis. On the contrary, if this estrogenic action occurs at an inappropriate time, or at doses not within physiological levels, it may cause adverse effects such as insulin resistance. This may happen when there is an exposure to an environmental estrogen, such as BPA, which causes postprandial hyperinsulinemia and insulin resistance in healthy male mice [START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF]. An excess of ER action mediated by an environmental estrogen such as BPA will provoke an increase in pancreatic -cell content and secretion, both in vivo and in vitro, overstimulating β-cells (Figure 2).

Excessive insulin signaling in the liver or endothelium produces dyslipidemia and in fat, it produces obesity, glucose intolerance, and dyslipedimia [START_REF] Biddinger | From mice to men: insights into the insulin resistance syndromes[END_REF]. In addition, an excessive insulin signaling may provoke insulin resistance in the liver and muscle [START_REF] Ueno | Regulation of insulin signalling by hyperinsulinaemia: role of IRS-1/2 serine phosphorylation and the mTOR/p70 S6K pathway[END_REF] as well as -cell exhaustion (Aston- [START_REF] Aston-Mourney | Too much of a good thing: why it is bad to stimulate the beta cell to secrete insulin[END_REF] and therefore, contributing to the development of type II diabetes (Figure 2).

Concluding remarks

Estrogen receptors are key molecules involved in blood glucose homeostasis.

Particularly, ER in β-cell has a crucial role in increasing proinsulin biosynthesis in response to physiological concentrations of E2. This may be an important mechanism for the islet of Langerhans to adapt to the high demand of insulin during pregnancy. This mechanism, however, can be activated as well in male mice by the widespread endocrine disruptor BPA, when there is no physiological need for an increase in insulin biosynthesis and secretion. Moreover, in vivo BPA treatment at environmentally relevant doses induces insulin resistance in healthy adult male mice. Is there a role for environmental estrogens such as BPA in the aetiology of type-2 diabetes? It has been demonstrated that type 2 diabetes only develops in insulin resistant subjects with the onset of β-cell dysfunction. Islet susceptibility to dysfunction depends on genetic and environmental factors, including intrauterine and early life environment [START_REF] Prentki | Islet beta cell failure in type 2 diabetes[END_REF]. The actions of environmental estrogens in uterus and early life on the function of the islet of Langerhans are still unknown and it is an important area of research to explore. In adult humans, however, epidemiological evidence exist that point to BPA as an important environmental risk factor in the development of type 2 diabetes [START_REF] Lang | Association of urinary bisphenol A concentration with medical disorders and laboratory abnormalities in adults[END_REF]. In addition, there are causal links between BPA exposure and insulin resistance [START_REF] Alonso-Magdalena | The estrogenic effect of bisphenol A disrupts pancreatic beta-cell function in vivo and induces insulin resistance[END_REF], alteration of insulin biosynthesis and secretion in β-cells in adult male mice [START_REF] Alonso-Magdalena | Pancreatic insulin content regulation by the estrogen receptor ER alpha[END_REF] and decrease of adiponectin in human adipocytes [START_REF] Hugo | Bisphenol A at Environmentally Relevant Doses Inhibits Adiponectin Release from Human Adipose Tissue Explants and Adipocytes[END_REF]. Insulin resistance and decrease of adiponectin should contribute to the development of type 2 diabetes, especially in subjects with a genetic susceptibility to β-cell failure. The BPA-induced increase in insulin biosynthesis and secretion produces overstimulation of -cells. This excessive demand, when prolonged in time, may produce endoplasmic reticulum stress, particularly when β-cell mass is not altered, contributing to -cell failure. Therefore, although the main causes of β-cell failure in type 2 diabetes are overnutrition and lack of exercise, environmental estrogens, in particular BPA, are strong candidates to exacerbate and accelerate the development of type 2 diabetes.

ACKOWLEDGMENTS

The author laboratories are funded by Ministerio de Ciencia e Innovacion grants BFU2005-01052, BFU2007-67607 and BFU2008-01492. CIBERDEM is an initiative of Instituto de Salud Carlos III. intolerance and insulin resistance. We postulate that the hyperinsulinemia generated by the excess or the absence of ER signaling, will produce dyslipidemia and obesity when affecting the liver and fat cells.

  -cells. The role of ER is still unknown, yet ER plays an important role in the regulation of insulin biosynthesis, insulin secretion and -cell survival. Activation of ER by 17-estradiol (E2) and the environmental estrogen Bisphenol-A (BPA) promotes an increase of insulin biosynthesis through a non-classical estrogen-activated pathway that involves phosphorylation of ERK1/2. The activation of ER by physiological concentrations of E2 may play an important role in the adaptation of the endocrine pancreas to pregnancy. However, if ER is over stimulated by an excess of E2 or the action of an environmental estrogen such as BPA, it will produce an excessive insulin signaling. This may provoke insulin resistance in the liver and muscle, as well as cell exhaustion and therefore, it may contribute to the development of type II diabetes.

Figure 1 .

 1 Figure 1. Proposed model for the action of 17β-estradiol and bisphenol-A (BPA) in

Figure 2 .

 2 Figure 2. Action of excessive insulin signaling in different tissues. Human studies


Dr Jason Peter Mansell
email: j.p.mansell@bris.ac.uk

David Farrar

Scott Jones

Maryam Nowghani

Cytoskeletal reorganisation, 125-dihydroxy vitamin D3 and human MG63 osteoblast maturation

Keywords: Osteoblasts, differentiation, cytoskeleton, calcitriol, mechanobiology

Bone tissue is especially receptive to physical stimulation and agents with the capacity to mimic the signalling incurred via mechanical loading on osteoblasts may find an application in a bone regenerative setting. Recently this laboratory revealed that the major serum lipid, lysophosphatidic acid (LPA), cooperated with 125dihydroxy vitamin D3 (D3) in stimulating human osteoblast maturation. Actin stress fiber accrual in LPA treated osteoblasts would have generated peripheral tension which in turn may have heightened the maturation response of these cells to D3. To test this hypothesis we examined if other agents known to trigger stress fiber accumulation cooperated with D3 in stimulating human osteoblast maturation.

Colchicine, nocodazole and LPA all co-operated with D3 to promote MG63 maturation in a MEK dependent manner. In contrast, calpeptin, a direct activator of Rho kinase and stress fiber accumulation did not act with D3 to secure MG63 differentiation. Herein we describe how the signalling elicited via microtubule disruption cooperates with D3 in the development of mature osteoblasts.

Introduction

Osteoblast differentiation in response to 125-dihydroxy vitamin D3 (D3) secures competent bone collagen mineralization [START_REF] Van Driel | Osteoblast differentiation and control by vitamin D and vitamin D metabolites[END_REF][START_REF] Van Driel | Evidence that both 1,25-dihydroxyvitamin D 3 and 24-hydroxylated D 3 enhance human osteoblast differentiation and mineralisation[END_REF]; D3 deficiency in childhood leads to rickets and an inadequate D3 status in adults results in osteomalacia [START_REF] Berry | Vitamin D metabolism, rickets and osteomalacia[END_REF]. Although it is widely recognised that D3 is essential for the provision of a mechanically sound mineralised skeleton the application of D3 to cultured osteoblasts in the absence of serum or selected growth factors does not drive their differentiation. We recently discovered that the principal serum factor co-operating with D3 in promoting human osteoblast maturation is lysophosphatidic acid [START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF]; in isolation D3 was unable to induce osteoblast differentiation but when used in conjunction with human serum a stark, synergistic maturation response occurred. We subsequently found that osteoblast maturation in response to D3 and serum could be blocked by Ki16425, a lysophosphatidic acid (LPA) receptor antagonist [START_REF] Ohta | Ki16425, a subtype-selective antagonist for EDG-family lysophosphatidic acid receptors[END_REF]. Furthermore the application of commercially available LPA in combination with D3 to MG63 osteoblasts led to striking, synergistic increases in both osteocalcin and alkaline phosphatase (ALP), proteins expressed by the differentiated phenotype [START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF].

Lysophosphatidic acid was identified as the serum borne agent responsible for triggering actin stress fibre accumulation in fibroblasts via activation of Rho [START_REF] Ridley | The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors[END_REF][START_REF] Ridley | Signal transduction pathways regulating Rho-mediated stress sibre formation: requirement for a tyrosine kinase[END_REF][START_REF] Ridley | Rho family proteins: coordinating cell responses[END_REF]. It has subsequently been established that LPA can actually elicit the same intracellular responses as generated by integrin-ECM contacts [START_REF] Hunger-Glaser | Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation[END_REF][START_REF] Bershadsky | Involvement of microtubules in the control of adhesion-dependent signal transduction[END_REF] A c c e p t e d M a n u s c r i p t 4 references therein), namely activation of Ras, focal adhesion kinase (FAK) and Rho.

Since these important discoveries LPA has found application in many studies aimed at enhancing our understanding of the mechanobiological events accompanying cytoskeletal reorganisation. For example, intracellular microrheological studies have recently reported how LPA enhances the viscosity and stiffness of the cytoplasm in serum starved Swiss 3T3 cells [START_REF] Kole | Rho kinase regulates the intracellular micromechanical response to adherent cells to Rho activation[END_REF]. Thus LPA could be thought of as a "mechanochemical" by virtue of its ability to both stimulate intracellular physicomechanical changes, via cytoskeletal reorganisation, and to mimic the signalling in response to integrin occupancy. In our previous report we postulated that events accompanying LPA-induced actin stress fibre accumulation somehow linked together with D3 in promoting osteoblast maturation [START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF]. Credence is given to this possibility because of the dependency on (osteoblast) mechanotransduction to the actin microfilaments of focal adhesion complexes [START_REF] Lipfert | Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125 FAK in platelets[END_REF][START_REF] Sinnett-Smith | Bombesin stimulation of p125 focal adhesion kinase tyrosine phosphorylation. Role of protein kinase C, Ca 2+ mobilization, and the actin cytoskeleton[END_REF][START_REF] Chen | Integrin mediated cell adhesion activates mitogen-activated protein kinases[END_REF][START_REF] Rankin | Platelet-derived growth factor modulation of focal adhesion kinase (p125 FAK ) and paxillin tyrosine phosphorylation in Swiss 3T3 cells. Bell-shaped dose response and cross-talk with bombesin[END_REF][START_REF] Seufferlein | Lysophosphatidic acid stimulates tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130. Signaling pathways and cross-talk with platelet-derived growth factor[END_REF][START_REF] Zhu | Integrin-dependent activation of MAP kinases: a link to shape-dependent cell proliferation[END_REF]. The actin component in turn acts in concert with the microtubular network in coordinating signal transduction emanating from focal adhesions. It has been proposed that the microtubules control integrin-dependent signalling and that the tension arising from the actin filaments might be under the control of microtubular assembly [START_REF] Bershadsky | Involvement of microtubules in the control of adhesion-dependent signal transduction[END_REF].

It is widely recognised that physical stimuli influences bone matrix metabolism and accrual (Liedert et al., 2006;Klein-Nulend et al., 2005;[START_REF] Harada | Control of osteoblast function and regulation of bone mass[END_REF]Pavalko et al., 2003 and references therein). Indeed Pavalko and colleagues (2003) remind us that "bending bone ultimately bends genes". Osteoblasts are in intimate A c c e p t e d M a n u s c r i p t 5 contact with the collagenous matrix they secrete via cell surface integrins. Physical stimuli result in changes in the extracellular environment, for example altered matrix orientation and/or initiation of strain and it is these changes that are detected by the integrins. On the cytoplasmic side of the osteoblast the integrin molecules make contact with the actin cytoskeletal network.

Thus the integrins are the mechanoreceptors relaying physical stimuli arising from alterations in the extracellular matrix (ECM) directly to the cytoskeleton [START_REF] Wang | Mechanotransduction across the cell surface and through the cytoskeleton[END_REF]. It is becoming clear that the function of mechanotransducing integrins is dependent on their linkage to the cytoskeleton [START_REF] Alenghat | Mechanotransduction: all signals point to cytoskeleton, matrix and integrins[END_REF].

One of our main goals is to identify novel D3-growth factor combinations for improving human osteoblast responses to bone implant materials. Both LPA and D3 co-operate in stimulating human osteoblast maturation, furthermore LPA exhibits many of the cell responses elicited via ECM-integrin contacts. The principle aim of this study therefore is to determine whether actin stress fiber accumulation per se provides a nexus with D3 in generating mature human osteoblasts and/or whether alterations in the microtubular component can fulfil D3 induced cellular differentiation. The information gleaned from such an investigation will assist in the identification of suitable small molecules for their subsequent application in a bone biomaterials context aimed at improving osseointegration. Novartis Pharma AG (Switzerland) and reconstituted to 5mM using DMSO.

MG63 cell culture

A c c e p t e d M a n u s c r i p t

Human osteoblast-like cells (MG63) were cultured in conventional tissue culture flasks (250 ml, Greiner) in a humidified atmosphere at 37ºC and 5% CO 2 . Cells were grown to confluence in DMEM/F12 nutrient mix supplemented with sodium pyruvate

(1 mM final concentration), L-glutamine (4 mM), streptomycin (100 ng/ml), penicillin (0.1 units/ml) and 10% v/v fetal calf serum. The growth media (500ml final volume) was also supplemented with 5ml of a 100 X stock of non-essential amino acids. Cells were grown to confluence and subsequently dispensed into 24well plates (Greiner, Frickenhausen, Germany) such that each well contained 1ml of a 2x10 4 cells/ml suspension (as assessed by haemocytometry). Cells were then cultured for 65 hours, the media removed and the cells treated with the same medium but lacking serum for 24 hours. In addition to the 24-well plate experiments cells were also seeded into 12-well plates for reporter assays, 60mm dishes or 4-well chamber slides for Erk phosphorylation and cytoskeletal studies respectively.

Co-treating MG63 cells with D3 and cytoskeletal reorganising agents -24 -well plate experiments

The establishment of MG63 cells for stimulations within 24 well plates was identical to that described above. These osteoblast-like cells were treated with 100ng/ml of either colchicine, nocodazole, taxol, thiocolchicoside or EIN in the presence or absence of 100nM D3. MG63 cells were also treated with calpeptin (5M) with or without D3. In some instances MG63 cells were treated with a combination of cytoskeletal agents and/or signal transduction inhibitors. For a positive control of MG63 maturation, cells were co-stimulated with D3 and LPA (20M) in the presence of 100g/ml FAFA. Cultures were left for a maximum of 72 hours prior to an A c c e p t e d M a n u s c r i p t 8 assessment of cellularity, osteocalcin production and alkaline phosphatase activity as we have detailed previously [START_REF] Yarram | Epidermal growth factor and calcitriol synergistically induce osteoblast maturation[END_REF][START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF] 2.4

Assessment of cell number

An assessment of cell number was performed using a combination of the tetrazolium

compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy-phenyl)-2-(4-
sulfophenyl)-2H-tetrazolium, inner salt (MTS, Promega, UK) and the electron coupling reagent phenazine methosulphate (PMS). Each compound was prepared separately in pre-warmed (37C) phenol red free DMEM/F12, allowed to dissolve and then combined so that 1 ml of a 1mg/ml solution of PMS was combined to 19 ml of a 2mg/ml solution of MTS. Immediately prior to treating cells with the MTS/PMS reagent, medium was aspirated from each of the wells and replaced with 0.5ml prewarmed (37C) phenol red free DMEM/F12. Each well was subsequently treated with 0.1ml of the MTS/PMS reagent mixture and the plates returned to the incubator for one hour. A blank consisted of media alone (0.5 ml) plus 0.1 ml of the MTS/PMS reagent mixture. Once incubated, samples (0.1 ml) from each well were dispensed onto a 96 well microtitre plate and the absorbances at 492 nm read using a multiplate reader. Plates were staggered to ensure that all samples were recovered for 96-well plating within 5 minutes to minimise any error introduced during the formation of further formazan product by the cell monolayer.

Measurement of alkaline phosphatase (ALP) activity

An assessment of ALP activity is reliably measured by the generation of pnitrophenol (p-NP) from p-nitrophenylphosphate (p-NPP) under alkaline conditions.

The treatment of cells to quantify ALP activity was similar to that described A c c e p t e d M a n u s c r i p t 9 previously [START_REF] Yarram | Epidermal growth factor and calcitriol synergistically induce osteoblast maturation[END_REF][START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF]. Briefly the remaining MTS/PMS reagent was removed and the monolayers rinsed with 1ml of phenol red free DMEM/F12 which was subsequently removed and the monolayers lysed with 0.1 ml of 25 mM sodium carbonate (pH 10.3), 0.1% (v/v) Triton X-100. After 2 min each well was treated with 0.2 ml of 15 mM p-NPP (di-tris salt, Sigma, UK) in 250 mM sodium carbonate (pH 10.3), 1.5 mM MgCl 2 . Lysates were then left under conventional cell culturing conditions for 1 hour. In order to confirm that the enzyme activity being measured was the bone/liver/kidney isoform, lysis buffers supplemented with either 1 mM levamisole or 1 mM phenylalanine were prepared.

After the incubation period, 0.1 ml aliquots were transferred to a 96 well microtitre plate and the absorbance read at 405 nm. An ascending series of p-NP (25-400 M)

prepared in the incubation buffer enabled quantification of product formation.

Osteocalcin (OC) enzyme linked immunosorbent assay (ELISA) -MG63 cells

were seeded into small flasks such that each flask contained 10ml of 20X10 3 cells/ml and the osteoblasts left for three days prior to medium removal and overnight starvation using serum free culture medium (SFCM). Once starved MG63s were either given fresh SFCM (supplemented with 250g/ml FAFA), D3 (100nM), LPA (20M), colchicine (100ng/ml), nocodazole (100ng/ml) or a combination of D3 with the aforementioned agents and the cells left to incubate for three days. After the treatment period conditioned media (20l) from each of the flasks were assayed for OC production using a commercial ELISA exactly as instructed by the manufacturer (immunodiagnosticsystems (ids), Boldon, Tyne & Wear, UK). Two independent experiments were performed using 4 replicates per treatment group and the data expressed as the mean osteocalcin concentration (ng/ml) +/-S.D.

A c c e p t e d M a n u s c r i p t 10

Smad-3 and activator protein-1 (AP-1) reporter assays

MG63 osteoblasts were seeded into 12-well plates such that 2ml of a 2x10 4 cells/ml was dispensed per well. Once the cells had reached approximately 60-70% confluence (typically within 24hr) the media was removed and replaced with serum free culture medium and the cells transiently transfected with 0.5g/well each of a Renilla control vector, pRL-TK-luc (Promega), and either 7AP-1-luc or CAGA 12 -luc (Smad-3 reporter) using Fugene6 transfection reagent (Roche). After 6 hours the cells were then treated with test reagents in serum free culture media and left for 18 hours prior to processing for luminometry using the Dual luciferase reagents as instructed (Promega). Luminescence was measured and the luciferase values normalised to control for transfection efficiency using the data obtained for the co-transfected control as previously described [START_REF] Davies | Induction of an epithelial-mesenchymal transition in human immortal and malignant keratinocytes by TGF-1 involves MAPK, Smad and AP-1 signalling pathways[END_REF].

Immunoblotting forSmad-2 phosphorylation

Briefly 5ml of a 5000 cell/ml suspension of MG63 cells were dispensed into 60mm dishes and left for 72hrs, the medium removed and replaced with serum free medium and the cells starved for 24hrs. The next day dishes were treated with 100ng/ml TGF (positive control), 10g/ml colchicine or vehicle for 1, 6 and 18 hours. After the stimulation period the media was removed, and the cells lysed using 0.1ml/dish of hot (~70°C) reducing buffer (3% SDS; 7% sucrose; 45mM Tris, pH 6.8; 35mM DTT; 0.01% bromophenol blue) and the samples subjected to SDS-PAGE and subsequent transfer onto PVDF. The membrane was then probed for phosphorylated Smad-2 using a rabbit polyclonal antibody (Cell Signalling Technologies, UK) and the secondary antibody was an anti-rabbit peroxidase conjugate (Sigma). The membrane 2.8 F-actin labelling using FITC-phalloidin.

Chamber slides, 4-well (Lab-Tek Chamber Slide systems, Nalge Nunc International, Roskilde, Denmark), were seeded with 1ml/well of a 5000 cells/ml suspension and the cells left as described above. After the initial culture period the medium was removed and the cells treated with either colchicine (100ng/ml), nocodazole (100ng/ml) or 5M calpeptin and the cells left for 24hrs. Following the incubation period cells were processed for F-actin detection using the fungal toxin phalloidin conjugated to FITC (Sigma, reconstituted to 0.5mg/ml in DMSO) exactly as described previously (Wulf et al., 1979). Briefly the culture media was removed and the cells rinsed once with PBS and then fixed for 5 minutes with 3.7% formaldehyde in PBS. The fixative was then aspirated and the cells treated with 0.2ml/well FITCphalloidin (5g/ml) in PBS for 40 minutes at room temperature. After the incubation period the cells were treated with DAPI slide mountant (Vectashield, Vector Laboratories, Peterborough, UK) and viewed under a DMLB fluorescence microscope (Leika, Bucks, UK) and images taken using Provia 400F 35mm film (Fuji).

Statistical analysis

Unless stated otherwise, all experiments described above were performed at least three times, each on separate days and using different passage numbers of cells. The minimum replicate number for each of the treatment groups and controls was four.

All data were subject to a one-way analysis of variance (ANOVA) to test for A c c e p t e d M a n u s c r i p t 13

Results

(i)

Both colchicine and nocodazole cooperate with D3 in stimulating MG63 maturation whereas calpeptin is without effect -The initial phase of this study was to ascertain if molecules reported to generate actin stress fibers could link with D3 in promoting human osteoblast maturation. To this end MG63 cells were treated with two structurally dissimilar microtubule disrupting agents or calpeptin, a direct activator of ROCK induced actin stress fiber accrual. Synergistic increases in ALP activity, and therefore osteoblast maturation, were observed for cells co-stimulated with D3 and either colchicine or nocodazole but not for MG63 cells treated with a combination of calpeptin and D3 (Fig. 1A). The differential ALP response was not attributed to altered cell numbers between the treatment groups (Fig. 1B) Confirmation of calpeptin bioactivity was supported by FITC-phalloidin stained actin stress fiber accrual of treated cells (Fig. 2B).

(ii) Osteocalcin (OC) expression in response to D3 is blunted by either LPA or microtubule disruption -Osteocalcin is expressed by committed osteoblasts but whether levels of this molecule are influenced by events surrounding cytoskeletal reorganisation have not been reported. As expected the treatment of MG63 cells with 100nM D3 led to a statistically significant increase (p<0.001) in OC production compared with vehicle controls (Fig. 3). However when MG63 cells were co-treated with D3 and colchicine/nocodazole/LPA the level of OC expression, although still greater than control cultures, was significantly attenuated (p<0.005). In each instance the co-stimulation of MG63 cells with D3 and LPA/colchicine/nocodazole lead to the expected synergistic increase in ALP activity compared to all other treatment groups (results not shown). and colchicine or LPA and D3 led to a stark increase in ALP activity. However when cells were exposed to 500nM TAE226, a FAK inhibitor, the differentiation response was not prevented. The application of higher concentrations of TAE226 (> 1M)

resulted in cell losses (data not shown).

A c c e p t e d M a n u s c r i p t

Discussion

Research conducted in our laboratory identified lysophosphatidic acid (LPA) as a potent mediator of D3 induced human osteoblast maturation [START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF]. At the time we postulated that LPA might influence osteoblast differentiation via its reported actions on the actin cytoskeleton [START_REF] Ridley | The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors[END_REF][START_REF] Ridley | Rho family proteins: coordinating cell responses[END_REF]Hall 1994, Ridley, 2001) and/or its ability to mirror the signalling initiated through ECMintegrin contacts [START_REF] Hunger-Glaser | Bombesin, lysophosphatidic acid, and epidermal growth factor rapidly stimulate focal adhesion kinase phosphorylation at Ser-910: requirement for ERK activation[END_REF][START_REF] Bershadsky | Involvement of microtubules in the control of adhesion-dependent signal transduction[END_REF]. Credence is given to either of these possibilities because of the important role of mechanical loading and the cytoskeleton in the regulation of bone mass [START_REF] Pavalko | A model for mechanotransduction in bone cells: the load bearing mechanosomes[END_REF]. To this end our primary focus was to ascertain whether other molecules reported to trigger actin stress fiber assembly and resultant cell tension could in some way link up with D3 in stimulating immature osteoblast differentiation. In parallel with LPA we screened the ability of three agents known to alter cytoskeletal reorganisation on their ability to cooperate with D3 in supporting human osteoblast maturation.

Calpeptin, colchicine and nocodazole are well known to stimulate actin stress fiber assembly. Originally developed as a cell permeable calpain inhibitor [START_REF] Tsujinaka | Synthesis of a new cell penetrating calpain inhibitor (calpeptin)[END_REF], calpeptin was serendipitously reported to stimulate actin stress fiber accumulation in fibroblasts via the inhibition of Shp-2 upstream of the small GTPase Rho [START_REF] Schoenwaelder | Evidence for a calpeptin-sensitive proteintyrosine phosphatase upstream of the small GTPase Rho. A novel role for the calpain inhibitor calpeptin in the inhibition of protein-tyrosine phosphatase[END_REF][START_REF] Schoenwaelder | The protein tyrosine phosphatase Shp-2 regulates RhoA activity[END_REF]. Both the naturally occurring alkaloid colchicine and the synthetic agent, nocodazole, both inhibit microtubule assembly and promote actin stress fiber accrual in a Rhodependent manner [START_REF] Bershadsky | Involvement of microtubules in the control of adhesion-dependent signal transduction[END_REF][START_REF] Enomoto | Microtubule disruption induces the formation of actin stress fibers and focal adhesions in cultured cells: possible involvement of the rho signal cascade[END_REF][START_REF] Zhang | Lysophosphatidic acid and microtubule-destabilizing agents stimulate fibronectin matrix assembly through Rhodependent actin stress fiber formation and cell contraction[END_REF][START_REF] Liu | Microtubule depolymerization induces stress fibers, focal adhesions and DNA synthesis via the GTP-binding protein Rho[END_REF]. As anticipated the co-treatment of MG63 osteoblast-like cells with Interestingly [START_REF] Gundersen | Microtubules and signal transduction[END_REF].

To ascertain whether the processes accompanying microtubule disruption cooperated with D3 in promoting increased ALP in osteoblasts we established two independent but complimentary approaches. In the first instance MG63 cells were treated with naturally occurring colchicine derivatives that have little or no impact on microtubule assembly; both colchiciene (EIN) and thiocolchicoside occur in the tissues and seeds of the Autumn Crocus, Colchicum autumnale. Although these agents exhibit, at most, a trivial action on the cytoskeleton they are biologically active and have proven efficacy in treating, for example, inflammatory, fibrogenic and hepatic diseases [START_REF] Chandrasekaran | Thiocolchicoside an anti-inflammatory analgesic agent with muscle relaxant action[END_REF][START_REF] Dvorak | Comparative effect of colchicine and colchiceine on cytotoxicity and CYP gene expression in primary human hepatocytes[END_REF]. In parallel with these compounds, MG63 cells were pre-treated with taxol, a microtubule stabilising drug, prior to their co-treatment with D3 and colchicine or D3 and nocodazole.

Neither thiocolchicoside nor EIN co-operated with D3 in stimulating an increase in ALP expression in osteoblasts. Similarly the pre-treatment of cells with taxol prevented the ability of both nocodazole and colchicine to act in concert with D3 in generating a maturation response. Collectively these data suggest that events accompanying the actual process of microtubule disruption in some way provide the nexus from which D3 is able to bolster a differentiation response in human osteoblasts.

During the course of this study, it became clear that colchicine was capable of eliciting many diverse effects upon mammalian cell behaviour. Although colchicine is A c c e p t e d M a n u s c r i p t 19 widely recognised to prevent microtubule assembly there are many studies reporting how this microtubule-disrupting agent influences a variety of signalling events in mammalian cells (Table 1). With regard to the signalling capabilities of colchicine, there are certainly two areas that deserve special attention in the context of D 3 induced MG63 maturation. First is the study by Dong and colleagues (2000) who reported increased Smad transcriptional activity in response to colchicine.

Transforming growth factor beta (TGF-) is one of the important factors for bone formation and remodelling [START_REF] Deng | Regulation of osteogenic differentiation during skeletal development[END_REF][START_REF] Zhang | Effects of transforming growth factorbeta 1(TGF-1) on in vivo mineralisation of human osteoblasts on implant materials[END_REF]. TGF- signalling is initiated by TGF- binding to the type II receptor, TRII, this interaction leads to the transphosphorylation of the TGF- type I receptor (TRI). Phosphorylated TRI in turn phosphorylates the Smad proteins that are in fact the transducers of the TGF- signalling cascade. It has been known for many years that a combination of D 3 and TGF- synergistically increases MG63 alkaline phosphatase activity [START_REF] Bonewald | Effects of combining transforming growth factor beta and 1,25-dihydroxyvitamin D 3 on differentiation of a human osteosarcoma (MG-63)[END_REF]. Consequently we examined whether colchicine could act as a TGF- mimetic by activating Smads.

The second noteworthy property of colchicine is its influence on the mitogen activated protein kinase (MAPK) pathway; in particular the activation (phosphorylation) of extracellular signal regulated kinase (Erk). This laboratory has already demonstrated the importance of Erk activation in D 3 induced osteoblast differentiation; co-stimulation of MG63 cells with epidermal growth factor and D 3 [START_REF] Yarram | Epidermal growth factor and calcitriol synergistically induce osteoblast maturation[END_REF] and the treatment of these cells with a combination of D 3 and LPA promotes osteoblast differentiation [START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF]. In both instances the maturation response could be completely blocked using U0126, an inhibitor of mitogen activated protein kinase kinase (MEK) and therefore Erk phosphorylation. et al., 1992). It is also known that nocodazole activates the small G protein Rho and stimulates tyrosine phosphorylation of focal adhesion kinase (FAK), paxcillin and Ras signalling pathways [START_REF] Vasquez | Nanomolar concentrations o nocodazole alter microtubule dynamic instability in vivo and in vitro[END_REF][START_REF] Wang | Microtubule-interfering agents activate c-Jun N-terminal kinase/stress-activated protein kinase through both Ras and apoptosis signalregulating kinase pathways[END_REF].

In Both colchicine and nocodazole appeared to activate MEK at the level of Raf in MG63 cells; the application of GW5074 blocked the ability of either colchicine or nocodazole and D 3 on MG63 maturation whereas FTI-277, a Ras inhibitor, resulted in a modest, yet statistically significant, attenuation of the differentiation response.

These findings are in support of those from Hayne and colleagues (2000) in which mitotic arrest to nocodazole requires Raf/MAPK activation. Similarly the data presented agree with other studies reporting Erk activation in response to colchicine [START_REF] Samarakoon | MEK/ERK pathway mediates cell shapedependent plasminogen activator inhibitor type 1 gene expression upon drug-induced disruption of the microfilament and microtubule networks[END_REF][START_REF] Schmid-Alliana | Microtubule integrity regulates src-like and extracellular signal-regulated kinase activities in human pro-monocytic cells. Importance for interleukin-1 production[END_REF]. This laboratory has previously reported that MEK activation is key to the maturation of MG63 cells cotreated with LPA and D3 [START_REF] Gidley | Lysophosphatidic acid cooperates with 1,25(OH) 2 D 3 in stimulating human MG63 osteoblast maturation[END_REF] and EGF and D 3 [START_REF] Yarram | Epidermal growth factor and calcitriol synergistically induce osteoblast maturation[END_REF].

It now appears that both colchicine and nocodazole also activate MEK in MG63 cells.

Part of our studies tackled whether FAK might also be implicated in the differentiation events generated in response to either colchicine or LPA. It is becoming clear that FAK is a component of the cellular mechanosensory apparatus in that it is able to detect and respond to local forces [START_REF] Tilghman | Focal adhesion kinase as a regulator of cell tension in the progression of cancer[END_REF]. In [START_REF] Halder | Therapeutic efficacy of a novel focal adhesion kinase inhibitor, TAE226, in ovarian carcinoma[END_REF][START_REF] Shi | A novel low molecular weight inhibitor of focal adhesion kinase, TAE226, inhibits glioma growth[END_REF]. However the application of 500nM TAE226 did not inhibit MG63 maturation in response to LPA/ colchicine and D 3 (data not shown). Although Bershadsky and colleagues (1996) reported phosphorylation of FAK in response to LPA and nocodazole in serum starved Swiss 3T3 cells it appears that FAK does not participate in the differentiation response of MG63 cells to D3 and cytoskeletal reorganising agents.

What remains unresolved is how does active MEK/Erk cooperate with D3 in driving a maturation response in osteoblasts? One possible explanation for the synergy observed for ALP in response to D3 and cytoskeletal reorganisation could be MEK dependent stimulation of activator protein-1 (AP-1). It is well known that the AP-1 family of transcription factors plays an important role in the development and maturation of osteoblasts [START_REF] Wagner | Functions of AP1 (Fos/Jun) in bone development[END_REF][START_REF] Marie | Transcription factors controlling osteoblastogenesis[END_REF]. With specific reference to the cytoskeleton and mechanotransduction it has emerged that the AP-1 component, c-Jun, is phosphorylated by colchicine-induced cytoskeletal reorganisation [START_REF] Lee | Cytoskeletal reorganization and TPA differently modify AP-1 to induce the urokinase-type plasminogen activator gene in LLC-PK 1 cells[END_REF]. Furthermore increased AP-1 binding activity in the promoter region of the human ALP gene has been reported for stretched periodontal ligament cells [START_REF] Peverali | Stretch-mediated activation of selective MAPK subtypes and potentiation of AP-1 binding in human osteoblastic cells[END_REF]. The application of 7AP-1-luc transfected osteoblasts revealed that cytoskeletal reorganisation precipitated by either colchicine or nocodazole does lead to activation of the AP-1 transcription factor complex. We hypothesise that the mechanism accounting for the synergistic increase in ALP activity in the models described is a consequence of two transcription factors acting at two different loci of Table 1. The impact of colchicine on mammalian cell physiology and signalling.

In addition to disrupting the assembly of microtubules and stimulating actin stress fiber accumulation it is becoming clear that colchicine is capable of activating and/or modulating diverse signalling systems in a variety of mammalian cells. Of particular relevance to osteoblast development and differentiation are the reports identifying colchicine as a stimulator of Smad transcriptional activity [START_REF] Dong | Microtubule binding to Smads may regulate TGF activity[END_REF] and Erk activation [START_REF] Schmid-Alliana | Microtubule integrity regulates src-like and extracellular signal-regulated kinase activities in human pro-monocytic cells. Importance for interleukin-1 production[END_REF]Samarakoon & Higgins 2003).

  culture reagents Tissue culture medium and fetal calf serum were obtained from Gibco (Paisley, Scotland) and essentially fatty acid free human serum albumin (FAFA) from Sigma (Poole, UK). Stocks of D3 (Sigma, Poole UK) were prepared in ethanol (100 M)and stored at 4ºC, LPA (Biomol, UK) was prepared in 1:1 ethanol:water (10mM) and stored at -20ºC and calpeptin (Calbiochem) reconstituted to 5mM in DMSO prior to storage at -20ºC. Colchicine (Sigma, Poole, UK) was dissolved in serum free culture medium to prepare a stock of 1mg/ml, filter sterilised and left refrigerated and both taxol and nocodazole (Sigma, Poole, UK) reconstituted in DMSO (1mg/ml) and stored at -20ºC. Thiocolchicoside was kindly provided by Alchem International (Faridabad, India) and prepared in ethanol to a working stock concentration of 100g/ml. Colchiceine (EIN) was prepared in house from colchicine exactly as described byBoyland and Mason (1938); briefly colchicine was prepared in 100mMHCl at a concentration of 100g/ml in a screw-cap microcentrifuge tube and placed in a boiling water bath for one hour. The resultant EIN preparation was immediately diluted 1000 fold in serum-free tissue culture medium and applied to MG63 cells.Inhibitors of Raf (GW5074) and MEK (UO126) were reconstituted to 10mM in DMSO whereas the Ras inhibitor (FTI-277) was prepared as a 10mM stock in serum free culture medium. All were obtained from Calbiochem and aliquots stored at -20ºC. The focal adhesion kinase (FAK) inhibitor, TAE226, was kindly provided by

  using a mouse monoclonal anti--tubulin (Sigma) to confirm equal protein loading.

  A c c e p t e d M a n u s c r i p t statistical significant. When a p value of <0.05 was found, a Tukey multiple comparisons post-test was performed between all groups.

  Events surrounding microtubule disruption are key to D3 induced osteoblast maturation -To determine if the signalling arising from microtubule disruption links up with D3 in generating mature osteoblasts these cells were treated with colchicine derivatives (EIN and thiocolchicoside) reported to have a trivial influence on tubulin dynamics. Furthermore cells treated with either colchicine or nocodazole were pretreated with the microtubule stabilising drug taxol. Both EIN and thiocolchicoside were unable to cooperate with D3 in generating mature MG63 cells(Fig 4A). Also, the pre-treatment of osteoblasts with taxol prevented the synergistic increase in ALP activity for those cells co-stimulated with D3 and the microtubule disrupting agents (Fig.4C). In each instance the very low ALP activities could not be explained by reduced cell numbers (Figs.4B & 4D).(iv)Evidence for the participation of Ras, Raf and MEK upon osteoblast maturation in response to microtubule disruption and D3 -We previously reported that osteoblast maturation in response to D3 and LPA could be blocked by UO126, an inhibitor of MEK. Similarly we also found that the same compound could neutralise the synergistic increase in ALP for MG63 cells co-treated with D3 and epidermal growth factor. The inclusion of UO126 to cells treated with both D3 and colchicine led to a marked inhibition of ALP activity (p<0.001, Fig.5A) The Ras inhibitor, FTI-277, led to a significant attenuation in ALP activity (p<0.01, Fig 5B)and the pretreatment of MG63 cells with GW5074, an inhibitor of Raf, led to a stark inhibition of ALP activity for cells co-stimulated with D3 and colchicine (p<0.001, Fig.5C). In each instance the very low or attenuated ALP activities could not be explained by reduced cell numbers (Fig.5D).

  Microtubule disruption in MG63 cells does not influence Smad 2 and 3 activity but does promote activator protein-1 activity -There are studies reporting on increased Smad transcriptional activity in response to microtubule disruption. To ascertain whether colchicine might influence osteoblast Smad 2 and 3 activity cells were stimulated with TGF and colchicine and appropriately prepared to detect phosphorylated Smad-2 by immunoblotting and Smad-3 activation via dual luciferase reporter assay respectively. As expected the treatment of MG63 cells with TGF resulted in a clear increase in phosphorylated Smad-2 at all treatment points whereas colchicine and vehicle treated groups were without effect (Figs 6A&B). Activator protein-1 (AP-1) plays a key role in bone development and homeostasis and it has been reported that colchicine can activate AP-1, albeit in non-bone cells. The application of 7AP-1-luc transfected MG63 cells reveals that the events associated with microtubule disruption do result in increased activity of AP-1 (p<0.01) in osteoblasts (Fig7) .(vi)Inhibition of FAK with TAE226 does not prevent osteoblast maturation -Physical stimuli are detected by FAK, but whether the events encompassing cytoskeletal reorganisation can influence osteoblast maturation via FAK activation has not been reported. As expected the co-stimulation of MG63 cells with either D3

  LPA and D3 led to a robust maturation response as supported by a clear, synergistic increase in ALP activity. Similar results were obtained for cells co-treated with both colchicine and D3 and nocodazole and D3 whereas calpeptin, either alone or in combination with D3, was without effect on ALP activity. Treatment of MG63 cells with calpeptin and the subsequent detection of F-actin accumulation via staining with FITC-phalloidin gave a clear indication of calpeptin bioactivity (Fig 2B).

  reported to stimulate Erk activation it was tempting to speculate that colchicine-mediated microtubule disassembly might induce the activation of the entire Ras-dependent cascade which in turn links up with D 3 in promoting MG63 maturation. Nocodazole, like colchicine impedes microtubule polymerisation(Jordan

  the first instance MG63 cells were pre-treated with an inhibitor of TRI which selectively neutralises the kinase activity of this receptor protein. As expected the inhibitor was able to prevent the increase in MG63 ALP activity for cells co-treated with D 3 and TGF(data not shown). When the inhibitor was applied to MG63 cells co-stimulated with either colchicine or nocodazole and D 3 the synergistic rise in ALP persisted. These findings indicated that microtubule disruption does not trigger a possible TGF mimetic response via TRI (data not shown). Osteoblasts were also probed for Smad2 phosphorylation in response to colchicine, nocodazole or TGF treatment. Whereas TGF led to Smad2 phosphorylation within 15 minutes there was no evidence for Smad2 phosphorylation in response to either colchicine or nocodazole. The final experiment in this particular series involved the application of a CAGA-12 Smad3 reporter assay. As anticipated the treatment of MG63 cells with TGF led to a striking increase in luciferase activity. However the application of either nocodazole or colchicine failed to generate a response. Thus far the findings would suggest that microtubule disruption does not, albeit for MG63 cells, generate a TGF mimetic event. Although there is evidence for raised Smad transcriptional activity in Mink lung epithelial cells experiencing microtubule disruption (Dong et A c c e p t e d M a n u s c r i p t 21 al., 2000) the conflicting findings for MG63 cells might be attributed to differences in adaptor and/or substrate molecules that participate in TGF signalling. In parallel with the studies aimed at investigating a potential TGF signalling response, a series of experiments were set up to explore whether microtubule disruption might activate MAPK pathways. To this end MG63 cells were initially pretreated with UO126, an inhibitor of MEK and resultant Erk phosphorylation. The inclusion of UO126 negated the influence of microtubule disruption and D 3 on MG63 differentiation. Thus colchicine and nocodazole were stimulating MEK activation.

  whether FAK might be implicated in the maturation response of MG63 cells to D 3 and LPA/colchicine we used TAE226. This recently developed low molecular weight inhibitor is well characterised as preventing FAK activation via blockade of tyrosine phosphorylation

  Figure & table legends

Figure 1 .

 1 Figure 1. Not all cytoskeletal reorganising agents cooperate with D3 in

Figure 2 .

 2 Figure 2. Calpeptin, colchicine and nocodazole trigger actin stress fiber

Figure 3 .

 3 Figure 3. Co-treating human osteoblasts with D3 and microtubule disrupting

Figure 4 .

 4 Figure 4. Events accompanying microtubule disruption interact with D3 in

Figure 5 .

 5 Figure 5. Osteoblast maturation in response to D3 and colchicine is dependent

Figure 6 .

 6 Figure 6. Cytoskeletal reorganising agents do not activate Smad 2 and Smad 3 in

Figure 7 .

 7 Figure 7. Nocodazole and colchicine activate activator protein-1 (AP-1) in human

  Fig. 1A

  Fig. 2

  Fig. 6A

Figure

  Figure

  Fig 7.

  the heightened expression of OC on receipt of D3 was significantly attenuated by LPA, colchicine or nocodazole. Although OC is expressed by committed osteoblasts it is well known that mice nullizygous for OC have no skeletal

	M a n u s c r i p t
	Thus agents with a propensity to raise ALP expression have far greater potential for promoting bone tissue mineralisation to those molecules which up-regulate OC production. With the exception of calpeptin the other factors reported to provoke actin stress fiber accumulation acted with D 3 in stimulating MG63 differentiation. Thus any signalling event(s) accompanying stress fiber accumulation per se appeared unsupportive of osteoblast maturation. What seemed more likely was that the A c c e p t e d
	signalling incurred by microtubule disruption in someway connected with D 3 in
	generating the mature osteoblast phenotype. Credence is given to such a possibility
	because signalling molecules interact with microtubules, furthermore drugs that

phenotype at birth but raised bone mass by six months of age

[START_REF] Ducy | Increased bone formation in osteocalcin-deficient mice[END_REF]

. Thus it is possible that a dampening of OC production by mature cells is associated with bone matrix accrual in adult life. Recently

[START_REF] Wolf | Energy regulation by the skeleton[END_REF]

has reviewed the importance of OC in energy metabolism rather than having a role to play in skeletal mineralisation. Suffice it to say ALP deficiency does result in hypophosphatasia, a skeletal abnormality characterised by an inadequately mineralised bone matrix

[START_REF] Whyte | Hypophosphatasia. In "The Metabolic and Molecular Basis of Inherited Disease[END_REF]

) whereas a paucity of OC is without influence on skeletal development.

18 influence microtubule stability are known to trigger and/or influence intracellular signalling cascades