Transforming growth factor-alpha gene expression and action in the seminiferous tubule: peritubular cell-Sertoli cell interactions

The local production and action of an epidermal growth factor (EGF)-like substance within the seminiferous tubule was investigated as a potential mediator of cell-cell interactions. Peritubular (myoid) and Sertoli cells were isolated and cultured under serum-free conditions. Proteins secreted by Sertoli and peritubular cells were found to contain a component that bound to the EGF receptor in a RRA. Separation of secreted proteins by reverse phase chromatography fractionated a protein that contained EGF bioactivity in its activity to stimulate growth of an EGF-dependent cell line. Biochemical properties examined for both Sertoli and peritubular cell EGF activities were similar with each other, but distinct from murine EGF. Northern blot analysis with an EGF cDNA probe did not detect EGF gene expression in peritubular, Sertoli, or germ cells. The possible production of an EGF-like substance such as transforming growth factor-alpha (TGF alpha) was investigated with a molecular probe to human TGF alpha. Both peritubular and Sertoli cells contained a 4.5-kilobase mRNA species that hybridized in a Northern blot analysis with a human TGF alpha cRNA probe. An immunoblot with a TGF alpha antisera confirmed the production of TGF alpha by the detection of a protein in both Sertoli and peritubular cell secreted proteins. TGF alpha gene expression was not detected in freshly isolated germ cells. Scatchard analysis revealed the presence of high affinity EGF receptors on peritubular cells and the absence of such receptors on Sertoli or germ cells. TGF alpha was found to stimulate peritubular cell proliferation, but had no effect on Sertoli cell growth. The effects of hormones and TGF alpha on Sertoli cell function and differentiation were assayed through an examination of transferrin production by Sertoli cells. TGF alpha had no direct effect on transferrin production or the ability of hormones to influence Sertoli cells. However, the presence of peritubular cells in coculture with Sertoli cells allowed TGF alpha to stimulate transferrin production. TGF alpha was also found to have relatively rapid effects on peritubular cell migration and the promotion of colony formation in culture. Cocultures of peritubular and Sertoli cells also responded to TGF alpha by the formation of large clusters of cells. Observations demonstrate the local production of TGF alpha by Sertoli and peritubular cells, and action of TGF alpha on peritubular cells and, potentially, Sertoli cells. The local production and action of TGF alpha may have a critical role as a paracrine/autocrine factor involved in the maintenance of testicular function.     

Actions of the testicular paracrine factor (P-Mod-S) on Sertoli cell transferrin secretion throughout pubertal development

Peritubular cells that surround the seminiferous tubules have been shown to produce a paracrine factor, termed P-Mod-S, that has dramatic effects on Sertoli cell function in vitro and is postulated to be important in the control of testicular function. The current study was designed to determine whether P-Mod-S has the ability to regulate Sertoli cell function during pubertal development. Sertoli cells were isolated from 10-, 20-, and 35-day-old rats which correspond to the prepubertal, mid-pubertal, and late pubertal stages of development. Histochemical analysis of cultured cells isolated from each age group was performed to establish the purity of the cell populations used. Testicular transferrin production by Sertoli cells was used as a marker of cellular differentiation. Basal production of transferrin by the cultured cells was found to increase during the pubertal period. P-Mod-S stimulated transferrin production by Sertoli cells isolated from 10-, 20-, and 35-day-old rats. FSH appears to enhance the ability of Sertoli cells to respond to P-Mod-S with cells obtained from 10-day-old rats. Sertoli cells from 35-day-old rats were nonresponsive to regulatory agents such as FSH. P-Mod-S alone, however, significantly stimulated transferrin production by Sertoli cells from this more adult stage of development. P-Mod-S was the only individual regulatory agent tested that could stimulate transferrin production by Sertoli cells from 35-day-old rats. Results indicate that P-Mod-S has the ability to regulate Sertoli cell function throughout pubertal development. Observations suggest that P-Mod-S and FSH may act together in the prepubertal testis to promote Sertoli cell differentiation and that P-Mod-S may act in the adult testis to maintain optimal Sertoli cell function and differentiation.     

Sertoli cell function varies along the seminiferous tubule: the proportion and response of transferrin secretors differ between stage-associated tubule segments

Recent studies from our laboratory demonstrated clearly that only a portion of all Sertoli cells secrete transferrin (TF). These findings raised the possibility that differences in the functional type of Sertoli cells from one location to another may account in part for the stage-related variation in TF release along the seminiferous tubule. In order to address this, Sertoli cells derived from tubule segments corresponding to stages III-V, VII, IX-XI, and XIII of the seminiferous epithelial cycle were subjected to reverse hemolytic plaque assays to determine whether the proportion of TF cells present in those segments were similar or different. We found 21.4 +/- 1.8%, 20.3 +/- 2.0%, 48.3 +/- 2.5%, and 49.2 +/- 3.2% of all cells secreted TF in III-V, VII, IX-XI, and XIII staged segments, respectively. Results obtained from immunocytochemical staining of cells from different sections agreed well with those obtained with plaque assays, indicating that we had detected most, if not all, TF cells in these cultures. In additional experiments, we found that cultured cells from stage III-V and VII responded to FSH or isoproterenol with a large increase in the rate of TF plaque formation, whereas cells from IX-XI and XIII segments appeared to be unaffected. In contrast, bovine fibroblast growth factor caused a marked increase in the rate of TF plaque formation with IX-XI cells and only a slight increase with cells from III-V staged segments. Thus, the manner in which Sertoli cells respond to several modulatory agents appears not only to be stage-dependent, but also to be specific to the agent in question. When taken together, our observations demonstrate that cultured TF secretors obtained from different staged segments of the seminiferous tubule differ in proportion and responsiveness. These findings, when viewed in light of reports of a constant number of Sertoli cells along the seminiferous tubule, suggest that Sertoli cells may acquire and lose the ability to secrete TF or respond to modulation as the seminiferous cycle progresses.     

A germ cell factor(s) modulates preproenkephalin gene expression in rat Sertoli cells.

Within the seminiferous tubule, both Sertoli and specific germ cells express opioid genes. Little is known about the paracrine regulation or role of opioid gene expression in the tubule. The present study shows that interactions among cells within the tubule may play a role in regulating preproenkephalin (PPenk) gene expression. Rat pachytene spermatocytes (PS) and round spermatids (RSd) were purified by centrifugal elutriation and established as primary cultures or co-cultured with Sertoli cells. The effects of germ cells or germ cell-conditioned media were studied to determine the expression of one of the opioid precursor genes in rat Sertoli cells, the PPenk gene. Following a 24 h co-culture with either PS or RSd, the expression of PPenk gene in Sertoli cells was increased 6.4- and 1.9-fold, respectively. Conditioned media obtained from either PS or RSd cultured for 20 h stimulated PPenk mRNA levels in Sertoli cells from as early as 2 h after exposure; maximum increases of 3.5- and 7.6-fold were observed at 12 h, respectively. The molecular weight of the germ cell factor(s) is greater than 30 kDa. 2 h after the addition of either PS- or RSd-conditioned media to Sertoli cells, small (2- to 2.6-fold, respectively) but significant (p less than 0.02) increases in extracellular cAMP levels were observed. Although both FSH and forskolin activated c-fos and PPenk gene expression in Sertoli cells, the germ cell factor(s) that stimulated PPenk mRNA levels did not affect the expression of this oncogene. These results indicate that germ cells interact with Sertoli cells, possibly by a protein(s) that acts as a short-loop paracrine factor, which regulates the expression of PPenk gene in Sertoli cells. These data suggest that stage-specific regulation of PPenk levels in Sertoli cells may occur in vivo.     

Pregnant mare serum gonadotrophin and follicle-stimulating hormone stimulation of cyclic AMP production in rat seminiferous tubule cells.

Pregnant mare serum gonadotrophin (PMSG) was found to stimulate the production of cyclic AMP in isolated rat seminiferous tubule cells. The PMSG was equipotent with highly purified ovine FSH. The subunits of PMSG were inactive in this system but recombination resulted in partial restoration of activity, and the combination of PMSG alpha-subunit with FSH beta-subunit was even more active. Desialation of PMSG, but not of ovine FSH, resulted in a reduction of slope and potency of this response, suggesting that sialic acid may be important for the interaction of PMSG and the Sertoli cell. Further tests of the responsiveness of this cell preparation demonstrated activity with several species of mammalian FSH molecules, but not with FSH molecules obtained from non-mammalian species.     

Basic fibroblast growth factor: production and growth stimulation in cultured adrenal cortex cells

Cultured bovine adrenal cortex cells express the basic fibroblast growth factor (bFGF) gene and contain, but under normal conditions apparently do not release, bFGF. However, once released, bFGF can stimulate proliferation of the cells, indicating that it could act as a self-stimulating growth factor for adrenal cortex cells. It is conceivable that the intracellular bFGF is released upon injury of the adrenal cortex and that it may be involved in the subsequent tissue repair mechanisms by stimulating the proliferation of adrenal cortical and vascular endothelial cells.     

Evaluation of plasma basic fibroblast growth factor (bFGF) in primary knee osteoarthritis patients

Aim of the work

The aim of this study was to investigate plasma basic fibroblast growth factor (bFGF) levels in patients with primary knee osteoarthritis (KOA) and to correlate it with physical performance, functional status and radiological severity.

Serum levels of the angiogenic cytokines basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) in multiple myeloma

Angiogenesis is a crucial process in growth and progression of cancer and there is growing evidence that neovascularisation is important in hematological malignancies. Since an increased angiogenic potential has been identified in multiple myeloma, we simultaneously measured circulating serum levels of the cytokines bFGF, VEGF, HGF and IL-6 by ELISA in 67 patients with multiple myeloma or monoclonal gammopathies of undetermined significance (MGUS) and in 20 controls. Median values of bFGF were 4.7 pg/ml in healthy volunteers, 6.2 in MGUS, 6.3 in myeloma stage I, 13.4 in stage II and 21.7 in stage III. Myeloma patients had significantly higher bFGF serum levels than controls (p<0.001). Pretreatment bFGF levels differed significantly in the Salmon and Durie stages I-III (p=0.02) and were significantly elevated in stage II-III compared to stage I myeloma (p=0.02). In patients responding to chemotherapy according to the CLMTF criteria, a significant decrease in serum bFGF, VEGF and HGF levels occurred (median pretreatment values for bFGF 23.9 pg/ml, post-treatment 6.5 pg/ml; p<0.001, for VEGF 223 pg/ml versus 105 pg/ml; p=0.02 and for HGF 1429 pg/ml versus 1077 pg/ml; p=0.02, respectively). In 11 patients who did not achieve a remission, there was no significant decrease in bFGF, VEGF and HGF levels. These data show that myeloma in stages II and III is associated with an increase in serum bFGF concentrations and give the first report that effective chemo-therapy is accompanied by a significant decrease in the angiogenic factors bFGF, VEGF and HGF, while no decrease of these factors could be found in nonresponders.     

Induction of c-fos, calcitonin gene expression, and acidic fibroblast growth factor production in a multipeptide-secreting neuroendocrine cell line

The multipeptide-secreting 44-2C cell line maintains differentiated function when grown in a serum-free, growth factor- and hormone-deprived milieu. The cells continue to synthesize and secrete calcitonin (CT), CT gene-related peptide, neurotensin, and somatostatin and respond to cellular secretagogues such as GRF and acidic and basic fibroblast growth factor. We designed experiments to ascertain the functional role(s) of cellular factors involved in the maintenance of the differentiated state in 44-2C cells. We report here the phenotypic transformation that occurs in these cells in the course of adjustment to the serum-free state. We also show the differential increase in CT-specific mRNA, the transient induction of c-fos, and the characterization of biologically active acidic fibroblast growth factor.     

Basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) are elevated in peripheral blood plasma of patients with chronic lymphocytic leukemia and decrease after intensive fludarabine-based treatment

Chronic lymphocytic leukemia (CLL) is characterized by extraordinary heterogeneity in terms of clinical course with overall survival ranging from several months to dozens of years. It is currently not possible to accurately predict the future clinical course in an individual patient. Angiogenesis has been recently reported as a potential prognostic factor in various hematological malignancies including CLL. The objective of the present study was to quantify plasma levels of key angiogenic activators vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) in patients with CLL and determine their potential change after intensive fludarabine-based treatment. Peripheral blood EDTA plasma concentrations of bFGF and VEGF were measured using comercially available enzyme-linked immunosorbent assay in 73 patients with untreated CLL (43 males, 30 females, median age, 65 years, range 31-88) and 80 healthy donors serving as control group. We found statistically significant increase in concentrations in patients with chronic lymphocytic leukemia compared to the control group (p < 0.0001 for both cytokines). No differences in angiogenic factors were noted between subgroups with low vs. intermediate vs. high-risk stage according to modified Rai staging or males vs. females. In twelve patients who achieved at least partial response after intensive fludarabine-based treatment, levels of bFGF as well as VEGF decreased significantly (bFGF, p = 0.0005; VEGF, p = 0.0068); in addition, they were no more significantly different from controls (bFGF, p = 0.524; VEGF, p = 0.728). Our data showed that key angiogenic activators bFGF and VEGF were elevated in plasma ofCLL patients. Furthemore, treatment with intensive fludarabine-containing regimens resulted in significant decrease of both cytokines. These data suggest that angiogenic cytokines may indeed play a significant role in CLL biology and that treatment with combination of fludarabine, cyclophosphamide +/- rituximab may exhibit antiangiogenic properties. Further studies with longer follow-up are necessary for evaluation of a possible association between angiogenic markers and progression-free survival or overall survival.     

Bcl-2 expression correlates positively with serum basic fibroblast growth factor (bFGF) and negatively with cellular vascular endothelial growth factor (VEGF) in patients with chronic lymphocytic leukaemia

A large proportion of B-chronic lymphocytic leukaemia (B-CLL) cells express the anti-apoptotic protein Bcl-2. Basic fibroblast growth factor (bFGF) has been shown to upregulate the expression of Bcl-2 in B-CLL cell lines. Vascular endothelial growth factor (VEGF) has been shown to enhance the survival of endothelial cells by upregulating the expression of Bcl-2. In the present study, we measured serum and cellular levels of bFGF and VEGF in 85 patients with CLL using a commercial quantitative sandwich enzyme immunoassay technique. Levels of Bcl-2 were also assayed concomitantly using Western blot analysis. The mean serum level of bFGF was 53.4 pg/ml (range 0-589) and that of VEGF 459.2 pg/ml (range 33-1793). The mean cellular level of bFGF was 158.3 pg/2 x 105 cells (range 0.8-841) and VEGF, 42.4 pg/2 x 105 cells (range 0-244). A high correlation was found between serum and cellular bFGF levels (P < 0.001), but not between the corresponding VEGF levels. Twenty-nine of 69 patients (42%) evaluated for Bcl-2 level, expressed it. The Bcl-2 level was positively correlated with the serum bFGF level (P = 0.007). However, surprisingly there was a negative correlation between Bcl-2 expression and intracellular VEGF level (P = 0.003). A positive correlation was also found between serum bFGF and disease follow-up time and log white blood cell count. These findings indicate that in CLL there is a correlation between angiogenesis-related factors and apoptosis-related protein expression, and elevated bFGF levels may account for the elevated Bcl-2 levels.     

Effects of basic fibroblast growth factor and nerve growth factor on lactate production, gamma-glutamyl transpeptidase and aromatase activities in cultured Sertoli cells

Sertoli cells are under the control of FSH and androgens and also respond to polypeptidic factors locally produced. Basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) have been proposed to belong to the large set of intratesticular regulators. The aim of the present investigation was to analyze the effects of bFGF and NGF on lactate production, gamma-glutamyl transpeptidase (gamma-GTP) and aromatase activities. Cultured Sertoli cells dose-dependently responded to bFGF by increasing lactate production and gamma-GTP activity under basal conditions. In FSH-stimulated cultures, a synergistic effect of FSH with bFGF for lactate production was observed. NGF did not produce changes in lactate production or gamma-GTP activity at any dose tested. Both peptides decreased FSH-stimulated aromatase activity. These results provide additional evidence for the participation of bFGF and NGF in the complex network of intratesticular regulators. bFGF has pleiotropic effects on Sertoli cell function while the actions of NGF seem to be more limited.     

Basic fibroblast growth factor: a potential mediator of stromal growth in the human prostate.

Studies were undertaken, using isolated prostatic epithelial and stromal cells, to evaluate the role of basic fibroblast growth factor (bFGF) in the regulation of benign prostatic growth. bFGF was detected in lysates, but not the conditioned media, of cultured prostatic epithelial and stromal cells by Western immunoblotting and immunoprecipitation of metabolically labeled proteins. Immunofluorescence analysis of benign human prostate localized the majority of bFGF to the prostatic stroma. In addition, bFGF was a potent stimulator of stromal cell proliferation in vitro, but was not mitogenic to cultured epithelial cells. Further studies demonstrated bFGF receptors (Kd = 258 pM; 61,400 receptors/cell) on stromal cells, but not epithelial cells. Epithelial cell-conditioned medium was mitogenic for stromal cells, suggesting the presence of paracrine interactions. However, bFGF does not appear to be the mediator of this interaction, since the mitogenic effect of epithelial cell-conditioned medium on stromal cells was not significantly reduced by the addition of anti-bFGF. Additional studies showed that concentrated stromal cell-conditioned medium was not mitogenic to cultured stromal cells under serum-free defined conditions, indicating the lack of an external autocine mechanism. These studies demonstrate that bFGF is actively synthesized by isolated prostatic epithelial and stromal cells, but is largely not secreted. Prostatic stroma, but not epithelia, are responsive to the mitogenic effect of bFGF in vitro. However, because of the limited secretion of bFGF by prostatic cells, the mechanism(s) of bFGF-mediated regulation of stromal growth remains unclear.     

Investigation of calmodulin and basic fibroblast growth factor (bFGF) in idiopathic myelofibrosis: evidence for a role of extracellular calmodulin in fibroblast proliferation

The urinary concentration of calmodulin and basic fibroblast growth factor (bFGF) was determined in a total of 53 patients with various chronic myeloproliferative disorders (CMPD), including 22 patients with idiopathic myelofibrosis (IMF). Calmodulin excretion was significantly elevated in IMF (0.29 ± 0.04 μg/mmol creatinine) ( P <0.001), when compared to polycythaemia vera (PV) (0.14 ± 0.02), essential thrombocythaemia (ET) (0.13 ± 0.04), chronic myeloid leukaemia (CML) (0.16 ± 0.02), unclassified myeloproliferative disorders (UMPD) (0.11 ± 0.02) and age-matched controls (0.1 ± 0.02) ( P <0.001). In contrast, bFGF was slightly elevated in all CMPD conditions when compared to age-matched controls. A neutralizing antibody to calmodulin was demonstrated to significantly influence the in vitro proliferation of normal human fibroblasts, an effect dependent on both cell density and the presence of fetal calf serum (FCS). Essentially, the antibody reduced FCS-induced proliferation of low-density fibroblasts but had little or no inhibitory effect on high-density fibroblasts in the absence of FCS. In addition, extracellular calmodulin was shown not to interact with known fibroblast mitogens, namely IFG-1, EGF, bFGF and PDGF. We conclude that extracellular calmodulin should be considered, in addition to PDGF, TFG-β and EGF, as a potential mitogen involved in the stromal reaction of idiopathic myelofibrosis.     

Basic fibroblast growth factor modulates the surface expression of effector cell molecules and primes respiratory burst activity in human neutrophils

Basic fibroblast growth factor (b-FGF) mediates a variety of biological responses such as angiogenesis and hematopoiesis. We examined the effect of b-FGF on human neutrophil functions in vitro. The surface expression of effector cell molecules on neutrophils was determined by flow cytometry and monoclonal antibodies. b-FGF increased the expression of CD11b leukocyte integrin and complement receptor type 1 on neutrophils and decreased the expression of L-selectin on neutrophils in a dose- and time-dependent manner. We also examined the effect of b-FGF on the respiratory burst activity in neutrophils. Although b-FGF alone did not induce intracellular oxidative product formation by neutrophils, it enhanced H(2)O(2) production in neutrophils stimulated by N-formyl-methionyl-leucyl-phenylalanine or phorbol myristate acetate. These findings suggest that b-FGF may participate in the inflammatory process via modulating the surface expression of effector cell molecules and enhancing respiratory burst activity in neutrophils.