The intracellular form of notch blocks transforming growth factor beta-mediated growth arrest in Mv1Lu epithelial cells

Notch signaling influences a variety of cell fate decisions during development, and constitutive activation of the pathway can provoke unbridled cell growth and cancer. The mechanisms by which Notch affects cell growth are not well established. We describe here a novel link between Notch and cell cycle control. We found that Mv1Lu epithelial cells harboring an oncogenic form of Notch (NICD) are resistant to the cell cycle-inhibitory effects of transforming growth factor beta (TGF-beta). NICD did not affect TGF-beta signaling per se but blocked induction of the Cdk inhibitor p15(INK4B). c-Myc, whose down-regulation by TGF-beta is required for p15(INK4B) induction, remained elevated in the NICD-expressing cells. c-Myc expression was also maintained in low serum, indicating that Notch's effects on c-Myc are not specific to TGF-beta. Our results are consistent with a model in which a strong Notch signal indirectly deregulates c-Myc expression and thereby renders Mv1Lu epithelial cells resistant to growth-inhibitory signals.     

Follicular stage-dependent tumor necrosis factor alpha-induced hen granulosa cell integrin production and survival in the presence of transforming growth factor alpha in vitro

The link between cell adhesion to extracellular matrix and integrin-mediated survival signals has been established in several physiological systems, and roles for the cytokines tumor necrosis factor alpha (TNF alpha) and transforming growth factor alpha (TGF alpha) have been suggested. TGF alpha stimulates fibronectin production in hen granulosa cells and is an important survival factor during follicular maturation. In contrast, the role of TNF alpha and its possible interaction with TGF alpha in the regulation of granulosa cell fate (death versus survival) during ovarian follicular development have not been fully elucidated. The object of the current study was to determine if TNF alpha and TGF alpha interact in the regulation of hen granulosa cell fibronectin and integrin content in the context of cell death and survival during follicular development. TGF alpha (0.1 or 10 ng/ml), but not TNF alpha (0.1 or 10 ng/ml), increased both cellular and secreted fibronectin content in granulosa cell cultures of F5,6 but not F1 follicles. The expression of integrin beta(3) subunit was also stimulated by TGF alpha in a follicular stage-dependent manner, and culture of F5,6 granulosa cells with TNF alpha in the presence of maximal stimulatory concentrations of TGF alpha potentiated this response. TGF alpha increased both F5,6 and F1 granulosa cell [(3)H]thymidine incorporation but not 3-(4,5-dimethylthiazol-2-yl)3,5-diphenyl tetrazolium bromide (MTT) metabolism. Although TNF alpha had no effect on [(3)H]thymidine incorporation irrespective of the presence of the growth factor, MTT metabolism was higher in F5,6 granulosa cells cultured for 24 h with both TNF alpha and TGF alpha than with either cytokine alone. Incubation of F5,6 granulosa cells for 48 and 72 h resulted in a TGF alpha-inhibited loss of cellular adhesion and detachment of granulosa cells from the growth surface. Although TNF alpha alone had no effect on cell morphology, it facilitated the reorganization of the granulosa cells into multicellular follicle-like structures in the presence of the growth factor. DNA degradation significantly increased between 0 and 72 h of culture in the absence of the cytokine but was suppressed by the addition of TGF alpha but not of TNF alpha. However, fluorometric analysis indicated that the primary type of cell death exhibited by F5,6 granulosa cells during extended culture and attenuated by the presence of TNF alpha and TGF alpha was necrosis and not apoptosis. The current study demonstrates that TNF alpha and TGF alpha interact in the regulation of granulosa cell integrin content and cell survival in vitro in a follicular stage-dependent manner. These findings suggest that follicular development is accompanied by a change in the intraovarian role of TNF alpha; it is atretogenic prior to follicular selection but prevents follicular demise during preovulatory growth.     

Insulin-like growth factor I supports differentiation of cultured osteoblast-like cells

Rat calvaria cells grown in culture for one week had properties of osteoblasts: a high content in alkaline phosphatase and a marked cyclic AMP response to parathyroid hormone (PTH). In short-term experiments, insulin-like growth factor I (IGF I) stimulated the incorporation of [14C] glucose into glycogen. When IGF I was present in the medium during 6 days the cell number increased slightly and there was a substantial, disproportionate rise in alkaline phosphatase activity of the cultures. Thus, IGF I stimulates growth, and in addition, and in contrast to other growth factors, mainly enhances differentiation of osteoblasts.     

Regulation of ovarian progestin production by epidermal growth factor in cultured rat granulosa cells

The modulation of ovarian steroidogenesis by epidermal growth factor (EGF) was investigated in cultured rat granulosa cells. Granulosa cells, obtained from ovaries of immature, hypophysectomized, estrogen-treated rats, were incubated for 2 days with EGF, follicle-stimulating hormone (FSH), or EGF plus FSH. Treatment with EGF did not affect estrogen production, but stimulated progestin (i.e. progesterone and 20 alpha-hydroxy-pregn-4-en-3-one) production in a dose-dependent manner. Stimulation of progestin production by EGF appears to be the result of an increase in pregnenolone biosynthesis as well as increases in the activities of 20 alpha-hydroxysteroid dehydrogenase and 3 beta-hydroxysteroid dehydrogenase/isomerase. Treatment with FSH increased both estrogen and progestin production by cultured granulosa cells. When cells were treated concomitantly with EGF, FSH-stimulated estrogen production was inhibited, while progestin production was further enhanced. The EGF enhancement of FSH-stimulated progestin production appears to be the result of synergistic increases in pregnenolone biosynthesis and 20 alpha-hydroxysteroid dehydrogenase activity, resulting in substantial increases in 20 alpha-hydroxypregn-4-en-3-one but not progesterone production. The effects of EGF were shown to be time-dependent. The concept of a direct action of EGF on rat granulosa cells is reinforced by the demonstration of high affinity (Kd approximately 3 X 10(-10) M), low capacity (approximately 5,000 sites/cell) EGF binding sites in these cells. Thus, EGF interacts with specific granulosa cell receptors to stimulate progestin but to inhibit estrogen biosynthesis.     

Fibroblast growth factor regulates the expression of luteinizing hormone receptors in cultured rat granulosa cells

We have investigated the effects of bFGF on both the FSH-induced LH receptor expression and cAMP production in cultured rat granulosa cells. Concentrations of pure FGF, from 10(-12) M to 10(-10) M, progressively inhibit the stimulatory actions of FSH with an ED50 of approximately 4 x 10(-12) M for both parameters. Higher FGF concentrations, from 4 x 10(-10) M to 10(-8) M, lead to a gradual reduction of the growth factor inhibitory effect. The effects of FGF are more prominent on the modulation of LH receptors than on the FSH-induced cAMP production. Moreover, FGF impairs the LH receptor formation induced by cholera toxin or 8-Bromo-cAMP, indicating that the growth factor also acts at a step distal to cAMP formation. The inhibitory effect of FGF on LH receptor expression increases during the entire course of granulosa cell differentiation, from 24 to 96 h, and is not due to variations in cell number or viability, but rather to a change in the content of LH receptors with no significant modification of binding affinity (KD congruent to 0.8 x 10(-10) M). These results suggest that bFGF may acutely regulate the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the ovarian follicular maturation.     

Epidermal growth factor regulation of connexin 43 in cultured granulosa cells from preantral rabbit follicles

Connexin 43 (Cx43), a gap junction protein expressed in differentiated granulosa cells, is necessary for normal follicular development. Cx43 expression and regulation by epidermal growth factor (EGF) were characterized in immature rabbit granulosa cells. Cx43 mRNA was expressed in the granulosa cells of primary follicles, but was undetectable in primordial follicles. Abundant expression of Cx43 mRNA was maintained in the granulosa cells of growing follicles through maturity. Granulosa cells were isolated from early preantral follicles and maintained in monolayer cultures for 72 hr. After the first 24 hr of culture, they were maintained for 48 hr in serum-free medium supplemented with 0, 1, 5, or 10 ng/ml of mouse EGF. Granulosa cell proteins were isolated, solubilized, and evaluated for Cx43 by Western blot analysis using antibodies to rat Cx43. Relative amounts of Cx43 protein (both phosphorylated and nonphosphorylated) were increased (P < 0.05) by EGF in a dose-dependent manner. Northern blot analysis of RNA from cultured granulosa cells demonstrated increased amounts of Cx43 mRNA in the EGF treated cultures (10 ng EGF/ml) relative to controls (P < 0.03). In summary, Cx43 gap junctions are synthesized in granulosa cells following the onset of folliculogenesis in vivo and their expression is enhanced by EGF in vitro.     

Tumor necrosis factor-alpha inhibits follicle-stimulating hormone-induced differentiation in cultured rat granulosa cells

We have investigated the effects of TNF-alpha on FSH-induced LH receptor expression, cAMP and progesterone production in cultured rat granulosa cells. TNF-alpha (0.5-100 ng/ml) inhibits the stimulating action of FSH on LH receptor formation in a dose-dependent manner with an IC50 of 1 ng/ml and an almost complete suppression of LH receptor induction for 50-100 ng/ml TNF-alpha. The inhibitory effect of TNF-alpha is not due to variations in cell number or viability but rather to a reduction of the LH receptor content per cell with no change in binding affinity (KD = 0.8 x 10(-10)M). TNF-alpha also inhibits the FSH-induced cAMP production but at a lower extent, with a maximum reduction of 60% for 100 ng/ml TNF-alpha. Moreover, TNF-alpha impairs the LH receptor formation induced by forskolin, cholera toxin or 8-Bromo-cAMP, indicating that the cytokine also acts at a step distal to FSH receptor and to cAMP formation. Finally, TNF-alpha decreases dramatically the progesterone synthesis that is stimulated by FSH, with a reduction to undetectable levels on and after 10 ng/ml TNF-alpha. These results suggest that TNF-alpha may drastically reduce the capacity of granulosa cells to differentiate upon FSH stimulation and to respond to LH during the physiological ovarian follicular maturation. Such anti-gonadotropic action of TNF-alpha on granulosa cell differentiation may be also relevant to the alteration of ovarian function during physiopathological processes like inflammatory or infection diseases.     

Modulation of adipocyte differentiation by tumor necrosis factor and transforming growth factor beta

Cultured TA1 adipocytes treated with tumor necrosis factor alpha (TNF) lose intracytoplasmic lipid and, over a period of days, come to resemble their predifferentiated progenitors (preadipocytes). To examine the extent to which this phenotypic reversion represents a return to a less differentiated cell, we examined three major characteristics that distinguish preadipocytes from adipocytes: (a) pattern of gene expression; (b) hormonal requirement for accelerated adipogenesis; and (c) pattern of protein synthesis. We found that within hours of TNF addition to adipocytes, mRNAs for genes whose expression is augmented during adipogenesis decreased to predifferentiated levels; in addition, like preadipocytes, TNF-treated adipocytes required exposure to hormones to accelerate adipogenesis. Further, the pattern of protein synthesis seen on polyacrylamide gels reverted to that seen before differentiation. Transforming growth factor-beta (TGF-beta) also caused a rapid decrease in expression of adipose genes when added to fully differentiated cells, an effect that was achieved by treatment with either TGF-beta 1 or TGF-beta 2. These effects were seen in the absence of a demonstrable proliferative response to either TNF or TGF-beta. Thus characteristics that define the "terminally" differentiated state in adipocytes are subject to modulation by environmental influences.     

Thrombospondin-1-dependent mechanism of transforming growth factor beta-1 activation in cultured human endothelial cells

Using two immortal human umbilical vein endothelial-derived cell lines we investigated the thrombospondin-1 (TSP-1)-dependent mechanism of the transforming growth factor beta-1 (TGFbeta1) activation. Exogenous human platelet TSP-1 was demonstrated to increase (1.7-2.0-folds) the concentration of active TGFbeta1 produced by both cell lines, but did not influence the concentration of total TGFbeta1. The process of TGFbeta1 activation is known to be regulated by G418GWSHW423 fragment of TSP-1 molecule. The simultaneous addition of TSP-1 and 100-fold molar excess (about TSP-1) of the synthetic GGWSHW peptide abolished TSP-1-induced TGFbeta1 activation. GGWSHW peptide inhibited the basal activation of TGFbeta1 in endothelial cells, whereas the secretion of total TGFbeta1 and TSP-1 was not altered. We conclude that TSP-1-dependent mechanism of endogenous TGFbeta1 activation is operation in human endothelial cells.     

Cellular distribution and relative amounts of vascular endothelium growth factor mRNA in granulosa cells from human preovulatory follicles.

Cells were obtained from patients undergoing in vitro fertilization. They were cultured and those producing vascular endothelium growth factor (VEGF) were detected by flow cytometry; relative amounts of mRNA were detected by RT-PCR and measured by PCR Elisa after RT-PCR products were biotinylated. Most of the granulosa cells produced VEGF. This production was maintained over 5 days in culture without adding hCG. The two diffusible forms, VEGF 121 and 165, were the most abundant. VEGF 145, which is specific to the reproductive system, was less abundant. VEGF 189, which is not freely secreted, was not produced by granulosa cells; small amounts were only detected in preparations containing leukocytes. TNF-alpha decreased VEGF production; the effect of TNF-alpha was neutralized by 10 nM staurosporine. Thus, the VEGF in human preovulatory follicles is mostly in the granulosa cells. These cells are therefore a major source of VEGF at ovulation and may play a key role in physiological and pathological processes which involve changes in vascular permeability and/or angiogenesis. The data also suggest that TNF-alpha via protein kinase C modulates the production of VEGF.     

Effects of bovine inhibin, transforming growth factor-beta and bovine Activin-A on granulosa cell differentiation

To determine whether inhibin and its related peptides might act locally to control granulosa cell function and differentiation, the dose- and time-dependent effects of bovine inhibin, the homo-dimer of the beta-chain of bovine inhibin (Activin-A) and porcine TGF beta on rat granulosa cell aromatase activity and progesterone synthesis were investigated in vitro. TGF beta enhanced FSH-induced aromatase activity and progesterone synthesis, and accelerated the peak response for progesterone synthesis. Activin-A on the other hand, augmented FSH-induced aromatase activity while arresting progesterone synthesis, and anti-luteinization effect. By contrast, exogenous inhibin had no detectable effect on the steroidogenic potential of these cells. Thus TGF beta and Activin, unlike their similar effects on the release of FSH by the pituitary, appear to affect ovarian granulosa cell function in different fashion, under conditions where inhibin itself has no effect.     

Regulation of extracellular matrix proteins by transforming growth factor beta1 in cultured pulmonary endothelial cells

Transforming growth factor beta-1 (TGF-beta1), which is present in lung tissue, has been suggested to play a role in modulating vascular cell function in vivo. The action of TGF-beta1 in vivo, especially at the local site of application to connective tissue, is anabolic and leads to pulmonary fibrosis and angiogenesis, strongly indicating that TGF-beta may have practical applications in repair of tissue injury caused by burns, trauma, or surgery. In the present study, we have used cultured bovine pulmonary artery endothelial (BPAE) cells as a model system. Expression of various proteins, including SPARC (secreted protein acidic and rich in cysteines), type IV procollagen and fibronectin (FN) was examined by radiolabeling the cells with [3H]proline, immunoprecipitation with specific antibodies, and Northern blot analyses by using specific cDNA probes. Cultured cells were labeled with [3H]proline for 24 h in either the absence or in the presence of TGF-beta1 (0-20 ng/ml). Incorporation of radioactivity was observed in a concentration-dependent manner, maximal at 5 ng/ml. Northern blot hybridization demonstrated that TGF-beta1 (5 ng/ml) treatment of BPAE cells caused an increase in steady-state levels     

Vascular endothelial growth factor stimulates osteoblastic differentiation of cultured human periosteal-derived cells expressing vascular endothelial growth factor receptors

Angiogenesis plays an important role in bone development and postnatal bone fracture repair. Vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptors (VEGFRs) are primarily involved in angiogenesis. This study investigated the expression of VEGF isoforms, VEGFR-1, and VEGFR-2 during the osteoblastic differentiation of cultured human periosteal-derived cells. In addition, the effect of exogenous VEGF on the osteoblastic differentiation of cultured human periosteal-derived cells was also examined. The expression of the VEGF isoforms (VEGF₁₂₁, VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆), VEGFR-1, and VEGFR-2 was observed in the periosteal-derived cells. Administration of KRN633, a VEGFR-1 and VEGFR-2 inhibitor, decreased the alkaline phosphatase (ALP) activity during the osteoblastic differentiation of cultured human periosteal-derived cells. However, the administration of VEGFR2 Kinase Inhibitor IV, a VEGFR-2 inhibitor, did not affect the ALP activity. The addition of recombinant human VEGF₁₆₅ elevated the ALP activity and increased the calcium content in the periosteal-derived cells. Treating the periosteal-derived cells with recombinant human VEGF₁₆₅ resulted in an increase in Runx2 transactivation in the periosteal-derived cells. These results suggest that exogenous VEGF stimulates the osteoblastic differentiation of cultured human periosteal-derived cells and VEGF might act as an autocrine growth factor for the osteoblastic differentiation of cultured human periosteal-derived cells.     

Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells

In this study, we test the hypothesis that the growth-promoting action of androgens on granulosa cells requires paracrine signaling from the oocyte. Mural granulosa cells (MGCs) from small antral (1-3 mm) prepubertal pig follicles were cultured in the presence or absence of denuded oocytes (DO) from the same follicles to determine whether mitogenic and/or steroidogenic responses, to combinations of FSH, insulin-like growth factor 1 (IGF1), and dihydrotestosterone (DHT) were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors we performed the same experiments, substituting growth differentiation factor 9 (GDF9), a known OSF, for the DO. OSFs and GDF9 both potently enhanced IGF1-stimulated proliferation, and inhibited FSH-stimulated progesterone secretion. Alone, DHT had little effect on DNA synthesis, but significantly enhanced the mitogenic effects of OSFs or GDF9 in the presence of IGF1. Denuded oocytes, GDF9, and DHT independently inhibited FSH-stimulated progesterone secretion, and androgen, together with DO or GDF9, caused the most potent steroidogenic inhibition. Focusing on mitogenic effects, we demonstrate that both natural androgen receptor (AR) agonists, testosterone and DHT, dose-dependently augmented the mitogenic activity of DO or GDF9. Antiandrogen (hydroxyflutamide) treatment, which is used to block androgen receptor activity, opposed the interaction between androgen and GDF9. In conclusion, androgens stimulate porcine MGC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9, via a mechanism that involves the AR. These signaling pathways are likely to be important regulators of folliculogenesis in vivo, and may contribute to the excess follicle growth that is observed in androgen-treated female animals.