Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and ...effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self-renewal or differentiation of hESCs.
Cell volume changes induced in various ways (anisosmotic environment, hormones, oxidative stress, substrate uptake) are an integral part of a signal transduction network regulating cell function. ...Cell swelling has received increasing attention as a stimulus for a variety of intracellular phenomena. One of the most remarkable effects of cell swelling is its powerful effect in inducing exocytosis of material in intracellular secretory vesicles. Secretion of essentially all so-packaged hormones including those from hypothalamus (thyrotropin-releasing hormone, TRH; gonadotropin-releasing hormone, GnRH), pituitary (LH, FSH, ACTH, MSH, TSH, prolactin, beta endorphin), pancreas (insulin, somatostatin, glucagon), heart (atrial natriuretic hormone) and kidney (renin) are stimulated in a concentration-related manner by medium hyposmolarity or isosmolar medium containing permeant molecules such as ethanol or urea (reviewed in Ref. 21). Cell swelling-induced exocytosis is not restricted to endocrine cells and hormones; medium hyposmolarity also induces secretion of exocrine pancreatic enzymes and myeloperoxidase from human polymorphonuclear leukocytes.
Cell volume changes induced in various ways (anisosmotic environment, hormones, oxidative stress, substrate uptake) are an integral part of a signal transduction network regulating cell function.1, ...2, 3 Cell swelling has received increasing attention as a stimulus for a variety of intracellular phenomena.4 One of the most remarkable effects of cell swelling is its powerful effect in inducing exocytosis of material in intracellular secretory vesicles. Secretion of essentially all so-packaged hormones5–29 including those from hypothalamus (thyrotropin-releasing hormone, TRH; gonadotropin-releasing hormone, GnRH), pituitary (LH, FSH, ACTH, MSH, TSH, prolactin, beta endorphin), pancreas (insulin, somatostatin, glucagon), heart (atrial natriuretic hormone) and kidney (renin) are stimulated in a concentration-related manner by medium hyposmolarity or isosmolar medium containing permeant molecules such as ethanol or urea (reviewed in Ref. 21). Cell swelling-induced exocytosis is not restricted to endocrine cells and hormones; medium hyposmolarity also induces secretion of exocrine pancreatic enzymes5 and myeloperoxidase from human polymorphonuclear leukocytes.25
Insulin-secreting pancreatic beta cells also express thyrotropin-releasing hormone (TRH). Although the physiological role of TRH in this localization is unclear, its participation in glucoregulation ...has been implied. To test this hypothesis, we blocked the last step of post-translational maturation of the TRH molecule by disulfiram, which is an active inhibitor of peptide alpha-amidation (PAM) within pancreatic islet cells. The treatment of male rats with 200 mg/kg/day of disulfiram during a 5-day period resulted in a low PAM activity, a high insulin content and its basal secretion from pancreatic islets, and the inability to release insulin in response to glucose (16.7 mM) or hypo-osmotic (30%) challenge in vitro. The addition of TRH (1 nM) to the medium during incubation restored the insulin content and both basal and glucose stimulated insulin secretions to control levels.
TRH plays an important role in the mechanism of insulin secretion and its response to glucose stimulation.
Cell swelling causes an immediate secretory response in various cell types. Induced secretion possesses some unique features suggesting the involvement of a specific signal transduction pathway. The ...effect of 10-20 microM GdCl3, 100 microM HgCl2, 1-100 microM indomethacin and 1-20 microM nordihydroguaiaretic acid (NDGA) on cell swelling-induced hormone secretion (isosmotic 80 mM ethanol or 15-30% hyposmotic medium) from incubated rat hypothalamic paraventricular nucleus (PVN) and posterior pituitary (oxytocin and TRH), isolated pancreatic islets (TRH) and perifused anterior pituitary cells (prolactin) were examined. To determine how general the effect of cell swelling is on exocytotic secretion, the release of two different neurohormones (thyrotropin releasing hormone -TRH and oxytocin) from the same tissue explant were studied. Both hyposmotic medium or isosmotic ethanol containing medium induced immediate TRH and prolactin release from the tested tissues. The effect of GdCl3, HgCl2, NDGA or indomethacin showed no inhibition of cell swelling induced secretion. In contrast to TRH, oxytocin release was not induced by isosmotic ethanol containing medium from the PVN or posterior pituitary.
These data indicate that signal transduction leading to exocytosis after cell swelling does not involve GdCl3 sensitive stretch activated channels, mercury sensitive aquaporins, or indomethacin and NDGA sensitive mediators including prostaglandins and leukotriens. Cell swelling-induced exocytosis possesses limited selectivity; cells specifically involved in water and salt regulation retain their specific response to osmotic stimuli.
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