Effect Of TGF-alpha Research Articles

イヌ卵子の体外成熟に及ぼすTGF-α，IGF-1およびPTEN阻害剤の影響

イヌ卵子の体外成熟に及ぼすTGF-α，IGF-1およびPTEN阻害剤の影響

P02.06 - Regulation of Ether À-Go-Go-1 Potassium Channel (EAG1) Gene Expression by Serum and TGF-Alpha in Human Liver Cancer Cells

Eag1 display oncogenic properties, and are proposed as markers and therapeutic cancer targets (1 - 5). Nevertheless, Eag1 regulation by endogenous factors is poorly understood. Because of the restricted distribution of Eag1 in normal tissues, we wondered whether serum regulates Eag1 expression. We also looked for ERK/MAPK activation and studied the effect of TGF-alpha (which activates ERK/MAPK) on Eag1 gene expression.

Transforming growth factor-alpha stimulates enterocyte proliferation and accelerates intestinal recovery following methotrexate-induced intestinal mucositis in a rat and a cell culture model

Recent evidence suggests that transforming growth factor-alpha (TGF-alpha) enhances enterocyte proliferation and exerts a gut trophic effect. The purpose of the present study was to evaluate the effect of TGF-alpha on enterocyte proliferation and intestinal recovery following methotrexate (MTX)-induced intestinal mucositis in rats and in Caco-2 cells. Nonpretreated Caco-2 cells and those pretreated with MTX were incubated with increasing concentrations of TGF-alpha. Cell proliferation was determined by FACS cytometry. Adult rats were divided into three groups: control rats treated with vehicle, MTX rats treated with one dose (20 microg/kg) of MTX given intraperitoneally, and MTX-TGF-alpha rats treated with one dose of MTX followed by two doses of TGF-alpha (75 microg/kg a day). Three days after MTX injection, rats were sacrificed. Intestinal mucosal damage (Park's score), mucosal structural changes, and enterocyte proliferation were measured at sacrifice. Western blotting was used to determine the level of extracellular signal-related kinase (ERK) protein, a marker of cell proliferation. A nonparametric Kruskal-Wallis ANOVA test was used for statistical analysis with P value less than 0.05 considered statistically significant. The in vitro experiment demonstrated that treatment with TGF-alpha of Caco-2 cells resulted in a significant stimulation of cell proliferation in a dose-dependent manner. The in vivo experiment showed that treatment with TGF-alpha resulted in a significant increase in bowel and mucosal weight, DNA and protein content in jejunum and ileum, villus height in jejunum and ileum, crypt depth in ileum, and increased cell proliferation in jejunum and ileum compared to the MTX group. MTX-TGF-alpha rats also had a significantly lower intestinal injury score in ileum when compared to MTX animals. The increase in levels of cell proliferation in MTX-TGF-alpha rats corresponded with the increase in ERK protein levels in intestinal mucosa. Treatment with TGF-alpha prevents mucosal injury, enhances ERK-induced enterocyte proliferation, and improves intestinal recovery following MTX-induced intestinal mucositis in rats. These findings correlated with the observation that TGF-alpha also caused a significant stimulation of cell proliferation in a Caco-2 cell culture model treated with MTX. These observations may have significant implications for the treatment of patients on chemotherapy who develop severe mucositis.

Effect of transforming growth factor-alpha on enterocyte apoptosis is correlated with EGF receptor expression along the villus-crypt axis during methotrexate-induced intestinal mucositis in a rat

The purpose of the present study was to evaluate the effect of transforming-growth factor-alpha (TGF-alpha) on enterocyte apoptosis following methotrexate (MTX) induced intestinal mucositis in a rat and in Caco-2 cells. Non-pretreated and pretreated with MTX Caco-2 cells were incubated with increasing concentrations of TGF-alpha. Cell apoptosis was determined by FACS cytometry. Adult rats were divided into four groups: Control, Control-TGF-alpha, MTX, and MTX- TGF-alpha rats. Three days later rats were sacrificed. Enterocyte apoptosis were measured at sacrifice. RT-PCR and Western Blotting was used to determine the level of Bax and Bcl-2 mRNA and protein. Real time PCR was used to measure epidermal growth factor receptor (EGFr) expression along the villus-crypt axis. The in vitro experiment has shown that treatment with TGF-alpha of Caco-2 cells results in a significant inhibition of cell apoptosis in a dose-dependent manner. In vivo experiment, a decreased levels of apoptosis in MTX- TGF-alpha rats corresponded with the decrease in Bax and with the increase in Bcl-2 at both mRNA and protein levels. The inhibiting effect of TGF-alpha on enterocyte apoptosis was strongly correlated with EGFr expression along the villus-crypt axis. In conclusion, treatment with TGF-alpha inhibits enterocyte apoptosis following MTX- injury in the rat.

TGF‐α induces upregulation and nuclear translocation of Hes1 in glioma cell

Both the Notch-signaling pathway and extracellular signal regulated kinase (ERK) cascade are involved in a wide variety of biological processes, such as proliferation, differentiation, survival, and tumorigenesis. Their dysregulation in recent studies have been shown to be associated with glioma formation. Here, we show that transforming growth factor-alpha (TGF-alpha) stimulated glioma cell line U251 growth and can partly compensate for the inhibitory effect of Notch-signaling inhibitor DAPT. The effect of TGF-alpha on ERK1/2 phosphorylation was prompt and transient and could be inhibited by mitogen-activated/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) specific inhibitor PD98059. Moreover, TGF-alpha was capable of up-regulating Hairy-enhancer of split1 (Hes1) expression which was independent of Notch1 activation, and of introducing Hes1 nuclear import in the presence of ERK1/2 activation. Collectively, our data suggest a potential linkage between ERK activation and the Notch-signaling pathway.

Responsiveness of intestinal epithelial cell turnover to TGF-α after bowel resection in a rat is correlated with EGF receptor expression along the villus–crypt axis

Recent evidence suggests that transforming growth factor alpha (TGF-alpha) enhances enterocyte proliferation and stimulates intestinal adaptation after massive bowel resection. In the present study, we evaluated the effects of TGF-alpha on enterocyte turnover and correlated it with epidermal-growth factor (EGF) receptor expression along the villus-crypt axis in a rat model of short bowel syndrome (SBS). Male rats were divided into three groups, sham rats underwent bowel transection (group A); SBS rats underwent a 75% bowel resection (group B); and SBS/TGF-alpha rats underwent bowel resection and were treated with TGF-alpha (75 microg/kg) (group C) from the seventh postoperative day. Parameters of intestinal adaptation, enterocyte proliferation and apoptosis were determined on day 15. Villus tips, lateral villi and crypts were separated using laser capture microdissection. EGF receptor expression for each compartment was assessed by quantitative real-time PCR (Taqman). Statistical analysis was performed using one-way ANOVA test, with P < 0.05 considered statistically significant. Treatment with TGF-alpha resulted in a significant increase in all parameters of intestinal adaptation. EGF receptor expression in crypts significantly increased in SBS rats (vs sham rats) (0.035 +/- 0.013 vs 0.010 +/- 0.002 Log ng Total RNA/18 s) and was accompanied by a significant increase in enterocyte proliferation (169 +/- 8 vs 138 +/- 5 BrdU positive cells/per 10 crypts, P < 0.05) and decreased apoptosis following TGF-alpha administration (group C). A significant decrease in EGF receptor expression at the tip of the villus (0.005 +/- 0.002 vs 0.029 +/- 0.014 Log ng Total RNA/18 s) and in the lateral villus (0.003 +/- 0.001 vs 0.028 +/- 0.006 Log ng Total RNA/18 s) in SBS (group B) rats (vs sham, group A) was accompanied by increased cell apoptosis in these compartments following treatment with TGF-alpha (group C). In a rat model of SBS, TGF-alpha increased enterocyte proliferation and stimulated intestinal adaptation. The effect of TGF-alpha on enterocyte turnover is correlated with EGF receptor expression along the villus-crypt axis.

Transforming growth factor α suppression of articular chondrocyte phenotype and Sox9 expression in a rat model of osteoarthritis

To define the roles of transforming growth factor alpha (TGFalpha) in cartilage degradation. Primary rat articular chondrocytes and articular osteochondral explants were cultured with TGFalpha to assess the effects of TGFalpha on chondrocyte physiology and phenotype. TGFalpha altered chondrocyte morphology through reorganization of the actin cytoskeleton and formation of stress fibers. Expression of anabolic genes, including aggrecan, type II collagen, and cartilage link protein, was reduced in response to TGFalpha. Proliferation of chondrocytes and formation of articular chondrocyte clusters was stimulated by TGFalpha. Expression of matrix metalloproteinase 13 and cathepsin C was increased by TGFalpha. We demonstrated the down-regulation of Sox9 messenger RNA and protein levels by TGFalpha. This was associated with reduced levels of phosphorylated and total SOX9 in cartilage explants upon TGFalpha treatment. In contrast, another growth factor identified in our microarrays, Kitl, had no effects on the chondrocyte parameters tested. To examine correlations between the increased levels of TGFalpha in experimental knee osteoarthritis (OA) with the levels of TGFalpha in humans with knee OA, a microarray analysis of mRNA from 13 normal and 12 late-stage OA cartilage samples was performed. Seven OA samples showed TGFA mRNA levels similar to those in the normal controls, but expression was markedly increased in the other 5 OA samples. These data confirm that TGFA transcript levels are increased in a subset of patients with OA. This study adds TGFalpha to the list of dysregulated cytokines present in degrading cartilage in OA. Since TGFalpha inhibits articular chondrocyte anabolic capacity, increases catabolic factors, and contributes to the development of chondrocyte clusters, TGFalpha may be a potential target for therapeutic strategies in the treatment of OA.

Transforming growth factor alpha acts as a gliatrophin for mouse and human astrocytes

Astrocyte death has been implicated in several neuropathological diseases, but the identification of molecules susceptible of promoting astrocyte survival has been elusive. We investigated whether transforming growth factor alpha (TGFalpha), an erbB1/EGFR ligand, which promotes glioma progression and affects astrocyte metabolism at embryonic and adult stages, regulates astrocyte survival. Primary serum-free astrocyte cultures from post-natal mouse and fetal human cortices were used. Transforming growth factor alpha protected both species of astrocytes from staurosporine-induced apoptosis. In serum-free medium, mouse astrocytes did not survive beyond 2 months while TGFalpha-treated astrocytes survived up to 12 months. Transforming growth factor alpha also promoted long-term survival of human astrocytes. We additionally extended TGFalpha proliferative effects to human astrocytes. After 3 days of permanent application, TGFalpha induced a major downregulation of both erbB1 and erbB2. This downregulation did not impair the functional activation of the receptors, as ascertained by their tyrosine phosphorylation and the continuous stimulation of both ERK/MAPK and PI3K/Akt pathways up to 7 days, the longest time examined. The full cellular effects of TGFalpha required activation of both transduction pathways. Enhanced proliferation and survival thus define TGFalpha as a gliatrophin for mammalian astrocytes. These results demonstrate that in normal, non-transformed astrocytes, sustained and functional erbBs activation is achieved without bypassing ligand-induced receptors downregulation.

Caveolin and GLT‐1 gene expression is reciprocally regulated in primary astrocytes: Association of GLT‐1 with non‐caveolar lipid rafts

Caveolae represent membrane microdomains acting as integrators of cellular signaling and functional processes. Caveolins are involved in the biogenesis of caveolae and regulate the activity of caveolae-associated proteins. Although caveolin proteins are found in the CNS, the regulation of caveolins in neural cells is poorly described. In the present study, we investigated different modes and mechanisms of caveolin gene regulation in primary rat astrocytes. We demonstrated that activation of cAMP-dependent signaling pathways led to a marked reduction in protein levels of caveolin-1/-2 in cortical astrocytes. Application of transforming growth factor-alpha (TGF-alpha) also resulted in a decrease of caveolin-1/-2 expression. Decreased caveolin protein levels were mirrored by diminished caveolin gene transcription. The repressive effect of TGF-alpha on caveolin-1 expression was MAP kinase-independent and partly mediated through the PI3-kinase pathway. Further downstream, inhibition of histone deacetylases abrogated TGF-alpha effects, suggesting that chromatin remodeling processes could contribute to caveolin-1 repression. Intriguingly, alterations of caveolin gene expression in response to cAMP or TGF-alpha coincided with reciprocal and brain-region specific changes in glial glutamate transporter GLT-1 expression. The reciprocal regulation of caveolin-1 and GLT-1 expression might be gated through a common PI3-kinase dependent pathway triggered by TGF-alpha. Finally, we showed that GLT-1 is located in non-caveolar lipid rafts of cortical astrocytes. In conclusion, this study highlights the occurrence of the reciprocal regulation of caveolin and GLT-1 expression during processes such as astrocyte differentiation via common signaling pathways. We also provide strong evidence that GLT-1 itself is concentrated in lipid rafts, inferring an important role for glial glutamate transporter function.

Extracellular glycosaminoglycan changes in healthy and overgrown gingiva fibroblasts after cyclosporin A and cytokine treatments.

It has been demonstrated that cyclosporin A (CyA) blocks the immune system, acts on cytoskeleton and stimulates the production of extracellular matrix (ECM) and transforming growth factor-beta1 (TGF-beta1). This cytokine, such as transforming growth factor-alpha (TGF-alpha), induces deposition of glycosaminoglycans (GAG), proteoglycans and collagen fibres in the ECM. In this work, we examined the effect induced by CyA, TGF-beta1 and TGF-alpha on cultures of healthy and overgrown human gingival fibroblasts in order to evaluate the glycosaminoglycan, cytoskeletal changes and the behaviour of fibroblasts after concanavalin A (Con A) treatment. Moreover, we examined gingival biopsies by Alcian blue histochemical staining and electron transmission microscopy. Total and extracellular sulphated GAG in overgrown gingiva specimens and in derived fibroblast cultures treated with CyA and cytokines were significantly higher than controls. The action of cytokines was increased (P < or = 0.01) compared with CyA with a greater effect of TGF-alpha in comparison with TGF-beta1; the electron microscopy showed ECM accumulation. The agglutinations showed the heterogeneity of fibroblast populations. Stimulation with Con A showed that the fibroblast population had cell surface heterogeneity, and could respond in a different way to both CyA and cytokine stimulus. Moreover, increased synthesis of GAG in overgrown gingiva compared with synthesis in normal fibroblasts before CyA treatment suggests a possible genetic origin of damage. As not all CyA-treated patients develop gingival overgrowth, a genetic predisposition may explain the different responses of gingival fibroblast populations.

Disparate effects of thyroid hormone on actions of epidermal growth factor and transforming growth factor-alpha are mediated by 3',5'-cyclic adenosine 5'-monophosphate-dependent protein kinase II.

Epidermal growth factor (EGF) and TGFalpha share the same plasma membrane receptor. In the present studies in HeLa cells, both EGF and TGFalpha caused MAPK (ERK1/2) activation and expression of the immediate-early gene c-fos. Thyroid hormone (T(4)) nongenomically enhanced EGF- and TGFalpha-induced MAPK activation. This T(4) action was duplicated by T(4)-agarose and blocked by tetraiodothyroacetic acid, which inhibits binding of T(4) to plasma membranes. TGFalpha-induced MAPK activation was potentiated by 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP) but not 8-chloro-cyclic adenosine monophosphate. TGFalpha, T(4), and 8-Br-cAMP each caused protein kinase A (PKA) II serine phosphorylation, whereas phosphorylation of PKA-II was not seen in cells treated with EGF or 8-chloro-cyclic adenosine monophosphate. In a PKA activity assay, the enzyme was stimulated by T(4), EGF, and TGFalpha; T(4) enhanced the effect of TGFalpha but not that of EGF. T(4), although it potentiated c-fos gene expression in EGF-treated cells, suppressed this effect in cells treated with TGFalpha. Cells exposed to 8-Br-cAMP also inhibited TGFalpha-stimulated c-fos expression. Studies of cell proliferation indicated that T(4) potentiated EGF action but inhibited that effect in TGFalpha-treated cells. The disparate effects of T(4) on actions of EGF and TGFalpha, which share the same cell surface receptor, are mediated by hormone phosphorylation and activation of PKA-II.

Transforming growth factor-alpha directly augments histidine decarboxylase and vesicular monoamine transporter 2 production in rat enterochromaffin-like cells.

For the production and vesicle storage of histamine, Enterochromaffin-like (ECL) cells express histidine decarboxylase (HDC) and vesicular monoamine transporter 2 (VMAT2). Although HDC and VMAT2 show dynamic changes during gastric ulcer healing, the control system of their expression has not been fully investigated. In the present study, we investigated the effect of transforming growth factor-alpha (TGF-alpha) and proinflammatory cytokines on HDC and VMAT2 expression in rat ECL cells. Time course changes in the expression of TGF-alpha during the healing of acetic acid-induced ulcers were studied. EGF receptor (EGFR) expression was also examined in ECL cells, whereas the direct effects of TGF-alpha and proinflammatory cytokines on HDC and VMAT2 expression in ECL cells were investigated using in vivo and in vitro models. During the process of ulcer healing, expression of TGF-alpha mRNA was markedly augmented. Furthermore, EGFR was identified in isolated ECL cells. TGF-alpha stimulated HDC and VMAT2 mRNA expression and protein production and also increased histamine release from ECL cells. Selective EGFR tyrosine kinase inhibitor tyrphostin AG1478 almost completely inhibited HDC and VMAT2 gene expression induced by TGF-alpha in vivo and in vitro. During gastric mucosal injury, TGF-alpha was found to stimulate ECL cell functions by increasing HDC and VMAT2 expression.

Transforming Growth Factor-α Attenuates N-Methyl-D-aspartic Acid Toxicity in Cortical Cultures by Preventing Protein Synthesis Inhibition through an Erk1/2-dependent Mechanism

Transforming growth factor-alpha (TGF-alpha), a ligand of the epidermal growth factor receptor, reduces the infarct size after focal cerebral ischemia in rat, but the molecular basis underlying the protection is unknown. Excitotoxicity and global inhibition of translation are acknowledged to contribute significantly to the ischemic damage. Here we studied whether TGF-alpha can rescue neurons from excitotoxicity in vitro and how it affects calcium homeostasis, protein synthesis, and the associated Akt and extracellular signal-regulated kinase 1/2 (Erk1/2) intracellular signaling pathways in mixed neuron-glia cortical cultures. We found that 100 ng/ml TGF-alpha attenuated neuronal cell death induced by a 30-min exposure to 35 microM N-methyl-D-aspartic acid (NMDA) (as it reduced lactate dehydrogenase release, propidium iodide staining, and caspase-3 activation) and decreased the elevation of intracellular Ca2+ elicited by NMDA. TGF-alpha induced a prompt and sustained phosphorylation of Erk1/2 and prevented the loss of Akt-P induced by NMDA 3 h after exposure. The protective effect of TGF-alpha was completely prevented by PD 98059, an inhibitor of the Erk1/2 pathway. Studies of incorporation of [3H]leucine into proteins showed that NMDA decreased the rate of protein synthesis, and TGF-alpha attenuated this effect. TGF-alpha stimulated the phosphorylation of the eukaryotic initiation factor 4E (eIF4E) but did not affect eIF2 alpha, two proteins involved in translation regulation. PD 98059 abrogated the TGF-alpha effect on eIF4E. Our data demonstrate that TGF-alpha exerts a neuroprotective action against NMDA toxicity, in which Erk1/2 activation plays a key role, and suggest that the underlying mechanisms involve recovery of translation inhibition, mediated at least in part by eIF4E phosphorylation.

Bcl-2 Expression Inhibits Liver Carcinogenesis and Delays the Development of Proliferating Foci

Tumor development is thought to require both increased proliferation and inhibition of apoptosis. However, the relationship between cell replication and cell death in liver tumorigenesis is complex because both proliferation and apoptosis increase during hepatocarcinogenesis. To investigate the effect of the anti-apoptotic gene Bcl-2 in liver carcinogenesis, we established a line of double transgenic mice that express transforming growth factor-alpha (TGF-alpha), a liver mitogen, and Bcl-2. Double transgenic mice, TGF-alpha and Bcl-2 single transgenics, and wild type received an injection of diethylnitrosamine at 15 days of age. This alkylating agent induces liver carcinogenesis and its effect is greatly enhanced by TGF-alpha. We report that Bcl-2 expression inhibited diethylnitrosamine-induced liver carcinogenesis and counteracted the enhancing effect of TGF-alpha. Bcl-2 delayed the growth of proliferative foci at the early stages of carcinogenesis and inhibited cell proliferation in these foci. The effect of Bcl-2 on liver carcinogenesis is consistent with its reported ability to interfere with cell replication. The data demonstrate that the expression of an anti-apoptotic gene during liver carcinogenesis causes a delay rather than an increase in tumorigenesis.

Comparison of the antisecretory and antiulcer activity of epidermal growth factor, urogastrone and transforming growth factor alpha and its derivative in rodents in vivo.

This study investigates the effects of epidermal growth factor (EGF), urogastrone (UG) and transforming growth factor-alpha (TGFalpha) and its derivative on dimaprit- and pentagastrin-induced gastric acid secretion and on acidified ethanol (AE)-evoked ulcer formation in anaesthetized rats. EGF, TGFalpha and UG administered subcutaneously (s.c.) 30 min before dimaprit inhibited gastric acid secretion. Against pentagastrin-stimulated secretion, TGFalpha inhibited, while EGF and UG potentiated, acid secretion dose-dependently. Intraduodenal (i.d.) administration of TGFalpha and UG had no effect, while EGF potentiated, both secretagogue-induced acid secretion in the same dosage schedule. Administration of either EGF, UG or TGFalpha i.v. bolus, in response to continuous infusion of dimaprit resulted in a significant (p < 0.05-p < 0.001) inhibition of acid secretion which was transient and returned to normal within 30-45 min for UG while it slowly returned to normal for EGF and TGFalpha. The truncated form of TGFa (amino acids 34-43) did not show any antisecretory effect when administered parenterally. Acidified ethanol produced gastric haemorrhagic lesions in the rat 1 h after oral administration. The gastric mucosal protective effects of TGFalpha, EGF and UG administered either orally or s.c. 30 min before the administration of AE were dose-dependent against this model of ulcer induction. Indomethacin (Indo), administered 15 min before AE to inhibit prostanoids biosynthesis, significantly (p < 0.001) reduced the cytoprotective effects of TGFalpha, EGF and UG and aggravated the ulcer index when administered s.c. The results show that PGs may be involved in mediating the protective effects of the three growth factors. Administration of NG-nitro-L argininemethylester (L-NAME) 15 min prior to TGFa, EGF and UG s.c. or orally, significantly (p < 0.001) decreased the degree of ulcer indices and was able to reduce the protective effects of TGFalpha, EGF and UG, thus including the role of NO in mediating the protective effects of these growth factors. In conclusion, these results have demonstrated that EGF, UG and TGFalpha have a short and reversible inhibitory effect on dimaprit-stimulated gastric acid secretion and each is effective parenterally but not orally. UG and EGF potentiated, while, TGFa inhibited pentagastrin-stimulated acid secretion. In addition, TGFalpha seems to lose its activity when it is truncated from the C terminus. The present study also suggests that EGF, UG and TGFalpha are equally effective against AE-induced gastric ulcer and bring about their cytoprotective action through their reduction of acid secretion and through PG and NO pathways.

Activation of the human transforming growth factor alpha (TGF-alpha) gene by the hepatitis B viral X protein (HBx) through AP-2 sites.

The HBx protein is known as a transactivator and potential oncogene, and TGF-alpha as a potent mitogen in hepatocellular carcinoma. By assays of serial deletion of the promoter of TGF-alpha gene and the cotransfection of HBx and AP-2 expression vectors, we observed that the HBx significantly activated the promoter activity through AP-2 sites located in the proximal region of the TGF-alpha promoter (-136 to -30). This effect was also observed in the heterologous promoter assay system containing AP-2 sites. The mutation analyses of three AP-2 sites in the promoter revealed that all three AP-2 sites contributed to the activation of the TGF-a gene in the presence of HBx. Accordingly, the mRNA level of TGF-alpha was significantly elevated in the HBx-expressing cell, HepG2-HBx and the HBV-producing cell, HepG2-K8. These results suggest that the HBx protein could increase the mitogenic effect of TGF-alpha by the transactivation of the gene through AP-2 binding sites and consequently, these interactions may accelerate the process of hepatocarcinogenesis.

Expression, action, and regulation of transforming growth factor alpha and epidermal growth factor receptor during embryonic and perinatal rat testis development.

The objective of the current study was to extend previous observations and examine the expression pattern and effects of transforming growth factor alpha (TGFalpha) and epidermal growth factor receptor (EGFR) on embryonic testis morphogenesis and growth. The expression of TGFalpha was determined after morphological sex determination (seminiferous cord formation at embryonic day 13 [ED13]) through perinatal testis development (postnatal day 5 [PD5]) with a quantitative reverse transcription-polymerase chain reaction procedure. Expression of messenger RNA (mRNA) for TGFalpha appeared to be more dynamic during testis development when compared with the expression of mRNA for EGFR. Message for TGFalpha was reduced at ED16 and PD4, and was elevated at PD0 during testis development. In contrast, EGFR mRNA levels were negligible at ED15 and were elevated constitutively from ED16 through PD5. Immunohistochemistry was conducted at ED14, ED16, ED19, PD0, PD3, and PD5 to localize cellular expression of both TGFalpha and EGFR. At ED16, positive staining for EGFR was localized to the cords, and by ED19, was mainly in the cords with slight expression in the interstitium. From PD0 to PD5, positive staining for EGFR was detected in the germ, Sertoli, and interstitial cells. Immunohistochemistry for TGFalpha detected localization at ED14 and ED16 to the Sertoli cells and to specific cells in the interstitium. From ED19 through PD5, TGFalpha was detected in the Sertoli, germ, and interstitial cells, and in endothelial cells within the interstitium. To determine the effects of TGFalpha on embryonic testis growth and seminiferous cord formation, ED13 testis organ cultures were treated with sense and antisense TGFalpha oligonucleotides. Antisense TGFalpha inhibited testis growth by 25%-30% in ED13 testis organ cultures when compared with sense oligonucleotide control pairs. To examine the effects of TGFalpha on perinatal testis growth, PD0 testis cultures were treated with different doses of TGFalpha. TGFalpha increased thymidine incorporation into DNA in PD0 testis cultures. Therefore, TGFalpha appears to have actions on both embryonic and perinatal testis growth. The regulation of TGFalpha and EGFR mRNA levels were examined using PD0 testis cultures treated with hormones that stimulate testis growth. Follicle-stimulating hormone (FSH) stimulated (P < .05) and testosterone tended to stimulate (P < .07) mRNA expression of EGFR. Epidermal growth factor stimulation of PD0 testis cultures did not affect levels of mRNA expression for EGFR, but did suppress expression of mRNA for TGFalpha. These results taken together demonstrate that TGFalpha can act to regulate early embryonic and perinatal testis growth. Furthermore, TGFalpha and EGFR expression can be regulated through growth stimulatory hormones such as FSH and testosterone.

Effects of transforming growth factors-alpha and -beta on proliferation and apoptosis of rat theca-interstitial cells.

Ovarian development, follicular growth and atresia require mechanisms regulating proliferation and death of ovarian cells including theca-interstitial (T-I) cells. Transforming growth factors-alpha and -beta (TGF-alpha and TGF-beta) are well recognized local modulators of T-I function. This study was performed to evaluate the effects of TGF-alpha and TGF-beta on ovarian T-I cell proliferation, differentiation and apoptosis. T-I cells from immature Sprague-Dawley rats were purified and incubated in chemically defined media. Proliferation was assessed by [3H]thymidine incorporation assay and by cell counting. Steroidogenically active cells were identified histochemically by detection of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity. DNA was extracted and apoptosis was identified by detection of internucleosomal DNA cleavage producing the characteristic 'ladder pattern' of low-molecular weight (LMW) DNA following agarose gel electrophoresis. Quantification of apoptosis was carried out with the aid of 3'-end labeling of DNA fragments with [32P]-dideoxy-ATP. TGF-alpha and TGF-beta stimulated [3H]thymidine incorporation by 2.2- to 3.1-fold and 1.7- to 3.4-fold respectively (P<0.005). A combination of TGF-alpha and TGF-beta produced a synergistic increase in DNA synthesis by 6.7-fold (at 1 ng/ml of each TGF-alpha and TGF-beta; P<0.001) and tenfold (at 10 ng/ml of each TGF-alpha and TGF-beta; P<0.001). Cell counting revealed that TGF-alpha increased the total number of cells 2.8-fold and TGF-beta 2.8-fold. The combination of TGF-alpha and TGF-beta increased the total cell count 3.2-fold, compared with control (P<0.05). The percentage of the steroidogenically active cells was 37+/-9% (mean+/-s.e.m. ) in the control cultures, 50+/-5% in the presence of TGF-alpha, 42+/-8% in the presence of TGF-beta, and 47+/-13% in the presence of both TGF-alpha and TGF-beta. TGF-alpha decreased apoptosis by 63+/-14% (P=0.02) while TGF-beta had no statistically significant effect. TGF-alpha in combination with TGF-beta produced the greatest inhibition of apoptosis by 73+/-8% (P=0.01). These findings demonstrate that TGF-alpha and -beta stimulate proliferation of both steroidogenically active and inactive T-I cells. Furthermore, TGF-alpha alone and in combination with TGF-beta protects T-I cells from apoptotic death. These effects of TGFs may be important in physiologic maintenance of ovarian mesenchymal growth and homeostasis as well as in pathophysiologic conditions associated with excessive growth of the T-I compartment, such as polycystic ovary syndrome.

A role for endogenous TGFalpha and associated signaling pathways in the differentiation of lens fiber cells.

TGFalpha is hypothesized to be an endogenous regulator of lens fiber terminal differentiation. With immunofluorescence, TGFalpha was localized to differentiating cells in the lens epithelium and superficial fiber cell mass of the adult chicken. A similar pattern of localization was also noted when differentiating epithelial cells were cultured. Immunoneutralization of endogenous TGFalpha inhibited the accumulation of filensin, a unique intermediate filament protein subunit restricted to developing vertebrate lens fibers. ELISA assays quantified the effects of TGFalpha on filensin expression. Surprisingly, inhibition of the TGFalpha receptors' tyrosine kinase activity with nanomolar concentrations of PD153035 increased the accumulation of differentiated characteristics in the presence or absence of ligand. Morphologically, PD153035-treated cells grew as aggregated masses and spread less well onto the substrate. Accompanying these morphologic changes was a complete inhibition of cell division. Post-receptor signaling events were examined with cAMP assays and Western blotting. TGFalpha did not affect cAMP levels while isoproterenol, an additional mediator of lens cell differentiation, caused significant increases in cAMP levels. Activation of ERK2 via dual phosphorylation was noted in response to TGFalpha but not isoproterenol. PD153035 reduced, but did not eliminate, ERK2 phosphorylation in response to TGFalpha. Phosphorylation of the CREB transcription factor was also observed in response to TGFalpha or isoproterenol. These data indicate that endogenous ligands can influence the expression of differentiated characteristics in cultured chick lens cells. A focus of multiple signaling pathways affecting filensin expression is the CREB transcription factor. While increased ERK2 activation may be involved in stimulating cell division, lower levels of persistent ERK2 activation promotes differentiation thus indicating some form of signaling pathway compartmentalization. This could also mean that additional receptors for TGFalpha not inhibited by PD153035 are responsible for promoting differentiation in chick lens cells.

Transforming Growth Factor-alpha Delays Gastric Emptying and Small Intestinal Transit in Suckling Rats

Transforming growth factor-alpha (TGF-alpha) is a biologically potent polypeptide detected in the gastrointestinal tract in suckling rats. The major goal of the present study was to test the hypothesis that the administration of TGF-alpha affects gastric emptying and small intestinal transit in suckling rats. Suckling (12-day-old) rats fasted 16 h received rat TGF-alpha subcutaneously (s.c.) or orogastrically in varying doses (0, 0.5, 1.0 microg/rat in 0.1% BSA). Control animals received 0.1% BSA only. Poly R-478 dye was used as a motility marker. Rats were decapitated 45 min after marker administration and the amount of dye in the stomach and the small intestine was measured by spectrophotometry. Subcutaneous administration of TGF-alpha significantly delayed stomach evacuation. In controls, the stomach contained 21.4 +/- 1.4% (mean +/- s(x)) of the Poly R-478 marker, whereas in TGF-alpha treated rats the stomach contained 37.2 +/- 2.8% of the total Poly R-478 dye given to animals. The delaying effect of TGF-alpha was time- and dose-dependent. Small intestinal transit was also significantly delayed. The proximal jejunum of TGF-alpha treated rats contained a 1.4-fold higher amount of marker in comparison with control rats. Orogastrically administered rTGF-alpha did not affect gastric emptying or intestinal transit. In conclusion, s.c. administration of rat TGF-alpha significantly delayed the gastrointestinal motility in vivo in suckling rats.