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M Keramidas

Publications and source records attributed to M Keramidas.

At least 19 recordsLinked to original sources
PubMed2005

Activation of transforming growth factor beta by Trypanosoma cruzi.

The anti-inflammatory cytokine, transforming growth factor beta (TGFbeta), plays an important role in Chagas disease, which is caused by the protozoan parasite Trypanosoma cruzi. In the current study, we show that the addition of an anti-TGFbeta antibody inhibited T. cruzi infection of cardiomyocytes, demonstrating the requirement for active endogenous TGFbeta. As TGFbeta is synthesized as a biologically inactive precursor, which is proteolytically processed to yield a mature, active homodimer, we hypothesized that T. cruzi could activate latent TGFbeta. To test this, we added recombinant latent TGFbeta to a TGFbeta-responsive reporter cell line in the presence of T. cruzi. We observed that T. cruzi was able to activate latent recombinant TGFbeta in this cellular model. We then investigated the ability of T. cruzi to activate latent TGFbetain vitro. We found that live T. cruzi, or cytosolic extracts of T. cruzi, activated latent TGFbeta in a dose- and temperature-dependent manner. The agent involved in TGFbeta activation was shown to be thermolabile and hydrophobic. Taken together, our studies demonstrate that T. cruzi directly activates latent TGFbeta. This activation is required for parasite entry into the mammalian cells and is likely to play an important role in modulating the outcome of T. cruzi infection.

Animals↗
PubMed2002

Increased Trypanosoma cruzi invasion and heart fibrosis associated with high transforming growth factor beta levels in mice deficient in alpha(2)-macroglobulin.

Trypanosoma cruzi proteinases are involved in host cell invasion in human patients and in mouse models. In mice, murine alpha(2)-macroglobulin (MAM) and murinoglobulin are circulating plasma proteinase inhibitors that also have important roles in inflammation and immune modulation. To define their role in experimental Chagas disease, we investigated the susceptibility to T. cruzi infection of mice that are deficient only in alpha2-macroglobulins (AM-KO) or in both MAM and monomeric murinoglobulin-1 (MM-KO), relative to the wild type (WT). Despite the high parasite load, parasitemia was lower in AM-KO and MM-KO mice than in WT mice. Nevertheless, we observed a significantly higher parasite load in the hearts of AM-KO and MM-KO mice, i.e., more amastigote nests and inflammatory infiltrates than in WT mice. This result demonstrates a protective role for MAM in the acute phase of murine T. cruzi infection. We further demonstrated in vitro that human alpha2-macroglobulins altered the trypomastigote morphology and motility in a dose-dependent way, and that also impaired T. cruzi invasion in cardiomyocytes. Finally, we demonstrated that the levels of transforming growth factor beta in AM-KO mice increased significantly in the third week postinfection, concomitant with high amastigote burden and important fibrosis. Combined, these in vivo and in vitro findings demonstrate that the MAM contribute to the resistance of mice to acute myocarditis induced by experimental T. cruzi infection.

Animals↗
PubMed2000

ACTH-regulated expression of vascular endothelial growth factor in the adult bovine adrenal cortex: a possible role in the maintenance of the microvasculature.

Endothelial cells lining vessels of endocrine tissues are fenestrated. Interactions with the local environment via either soluble factors or cell-cell interactions appear to govern this terminal endothelial differentiation. Adrenocorticotropin (ACTH) has previously been reported to modulate endothelial fenestration in the rat adrenal cortex. Since vascular endothelial growth factor (VEGF) has been characterized as a potent inducer of endothelial fenestration, we aimed to characterize the status of VEGF expression in the bovine adult adrenal cortex and asked whether ACTH may regulate VEGF expression. By immunohistochemical analysis, we observed VEGF expression in steroidogenic cells from both zona glomerulosa and zona fasciculata of the bovine adrenal cortex. Double-labeling experiments performed on isolated cells in primary culture revealed VEGF immunoreactivity, essentially colocalized with the Golgi apparatus. The expression of two predominant VEGF isoforms, VEGF(121) and VEGF(165), was observed by RT-PCR analysis. ACTH (10 nM) was found to rapidly (within 2-4 h) increase the abundance of these VEGF transcripts, as assessed by both RT-PCR and Northern blot analysis. In parallel, ACTH significantly induced VEGF secretion into the medium of fasciculata cells in primary culture. Thus, our data are consistent with the involvement of ACTH, through its regulation of VEGF expression, in the maintenance of the adult adrenal cortex endothelium.

Adrenal Cortex↗
PubMed2000

Transforming growth factor-beta receptor types I and II in cultured porcine leydig cells: expression and hormonal regulation.

The steroidogenic activity of testicular Leydig cells is controlled both by the pituitary hormone (LH) and by growth factors such as transforming growth factor-beta peptides (TGFbeta1, -2, and -3; inhibin/activin; and anti-Mullerian hormone). By using primary cultures of porcine Leydig cells as a model, the aim of the study was to identify and characterize the TGFbeta receptors and to study their regulation by LH/hCG. TGFbeta receptors have been identified and characterized through three different approaches, including cross-linking experiments and Western and Northern blotting analyses. In cross-linking experiments, labeled TGFbeta was shown to bind to three different molecular species of 300, 80, and 53 kDa, which may correspond to the protein betaglycan (also known as TGFbeta type III receptor) and TGFbeta type II and I receptors (TGFbetaRII and TGFbetaRI), respectively. The presence of TGFbetaRI and -RII was further demonstrated by Western blotting analysis using specific polyclonal antibodies. Finally, the expression of betaglycan, TGFbetaRII, and TGFbetaRI messenger RNAs, was confirmed by Northern blotting analysis, as shown by the presence of 6.4-, 4.6-, and 5.8-kb messenger RNAs, respectively. By using a RT-PCR approach, the mediators of the TGFbeta signal, Smads 1-7, were also detected in cultured Leydig cells. TGFbetaRI and TGFbetaRII protein levels were enhanced by hCG/LH in a dose-dependent (maximal effect with 0.3 ng/ml hCG) and time-dependent (maximal effect observed after 48 h of hCG treatment) manner. Furthermore, to determine whether the stimulatory effect of LH/hCG was mediated by testosterone, use was made of aminogluthetimide, an inhibitor of cytochrome P450scc. The inhibition oftestosterone formation did not affect the stimulatory effect of LH/hCG on TGFbetaRI and -RII levels, suggesting that the gonadotropin action is not mediated by the steroid hormone. Together, the present findings demonstrate that the TGFbeta receptors are expressed and are under hormonal (gonadotropin) control in cultured porcine Leydig cells.

Aminoglutethimide↗
PubMed2000

Paracrine control of the adult adrenal cortex vasculature by vascular endothelial growth factor.

The extensive vascular network that irrigates the adult adrenal cortex is essential for both the delivery of oxygen and nutrients to glandular steroidogenic cells and for the rapid and efficient export of corticosteroid products from these cells into the blood flow. During experimental and pathological changes in adrenal cortex size caused by ACTH overproduction or deficiency, the vasculature evolves in a coordinated manner with the mass of glandular cells so that blood vessel formation/regression and cortical gowth/atrophy are respectively synchronized. In addition to its previously reported expression in human fetal adrenocortical cells, the angiogenic factor VEGF-A appears also to be strongly expressed by both glomerulosa and fasciculata cells of the adult bovine adrenal cortex, when the endothelium is quiescent. Moreover, the expression of the two major transcripts encoding the 121 and the 165 amino acid-long isoforms of VEGF-A was observed to be rapidly (within 2-4 h) up-regulated (2-3 fold) by ACTH in primary cultures of bovine fasciculata and glomerulosa cells. The expression of the signalling VEGF receptors R1 (flt-1) and R2 (flk-1) was restricted to the endothelial cells of the cortex whereas neuropilin-1 was expressed by both endothelial and steroidogenic cells. This suggests that, under the control of the pituitary hormone ACTH, VEGF exerts a paracrine control over the vasculature of the adult adrenal cortex. Given its known effects as an anti-apoptotic agent and an inducer of endothelial fenestration, VEGF is likely to play a role in the maintenance of the dense and fenestrated vascular bed of the adrenal cortex. The vasculature thus appears as an important secondary target of ACTH action in the physiological control of adrenal cortex homeostasis.

Adrenal Cortex↗
PubMed1999

Tissue inhibitor of metalloproteinase-2 (TIMP-2) expression is strongly induced by ACTH in adrenocortical cells.

Besides its acute and chronic effects on corticosteroid synthesis, the pituitary adrenocorticotropic hormone (ACTH) regulates diverse adrenocortical biological functions including the synthesis of a number of mitochondrial, cytoplasmic, and secreted proteins. ACTH-induced secreted proteins are candidates to act as local extracellular relays of the hormone in either an autocrine or a paracrine manner. In the present study, we report that stimulation of primary cultures of bovine adrenocortical (BAC) fasciculata cells with 10 nM ACTH for 24 h results in a mean 8 +/- 4-fold induction of the synthesis of a secreted protein presenting an apparent Mr of 21 kDa. Peptide microsequencing and Western blotting allowed us to identify this 21-kDa ACTH-induced protein as the tissue inhibitor of metalloproteinase-2 (TIMP-2). The induction of TIMP-2 by ACTH required transcription, was mimicked by 8-bromo cyclic 3'-5' adenosine monophosphate, but was not observed in response to angiotensin II, IGF-1, fibroblast growth factor-2, transforming growth factor-beta1, or cortisol treatments. ACTH stimulated TIMP-2 mRNA levels by a factor 4, whereas TIMP-1 mRNA levels were not affected and TIMP-3 mRNA remained undetectable. The biological activity of TIMP-2 varied accordingly, as we observed that the conditioned medium of ACTH-treated BAC cells was four times more potent at inhibiting gelatinolytic activity than was the conditioned medium of control cells. Because the proteolytic activity of both progelatinase-B and progelatinase-A secreted by BAC cells remained latent, whether in the presence or in the absence of ACTH, a paracrine rather than autocrine role is proposed for TIMP-2 in the adrenal cortex.

Adrenal Cortex↗
PubMed1999

Bovine thrombospondin-2: complete complementary deoxyribonucleic acid sequence and immunolocalization in the external zones of the adrenal cortex.

Given the variety of biological functions in the adrenal cortex that are controlled by ACTH, we hypothesized that some extracellular proteins act as biological relays for this systemic hormone. One candidate protein [corticotropin-induced secreted protein (CISP)] was purified from the conditioned medium of bovine adrenocortical cells on the basis of a 5- to 14-fold increase in its synthesis after the addition of ACTH. We report here the cloning of overlapping complementary DNAs that span the sequence encoding the full-length protein (1170 amino acids). The deduced CISP protein sequence is 89% identical to that of human thrombospondin-2 (TSP2), but only 61% identical to that of bovine TSP1, confirming that CISP is the bovine ortholog of TSP2. The bovine TSP2 sequence aligned perfectly with human, mouse, and chicken TSP2 sequences, except for a gap of 2 amino acids located in a linker region. All 58 cysteine residues that are conserved in other species were present in the bovine sequence as well as most of the functional domains. Most endocrine tissues (adrenal cortex, testis, ovary, and placenta) appeared to express TSP2, as determined by Western blot analysis. The highest levels of TSP2 protein were found in the adrenal cortex, followed by the heart, spleen, brain, and kidney. A differential extent of N-glycosylation or tissular proteolytic maturation may be responsible for the mol wt differences observed between bovine TSP2 detected in the medium from primary cultures and that in fresh tissue extracts. The immunohistochemical analysis of the distribution of TSP2 in the bovine adrenal gland revealed that the protein is much more abundant in the external zones (zona glomerulosa and zona fasciculata) than in the internal reticularis zone, a pattern similar to that reported for ACTH receptors. This distribution clearly suggests that TSP2 is a candidate relay protein for a subset of ACTH actions in the adrenal cortex.

Adrenal Cortex↗
PubMed1997

Expression of laminin and its possible role in adrenal cortex homeostasis.

The adult mammalian adrenal cortex undergoes permanent regeneration. This process implies a cellular proliferation step restricted to the external zone of the tissue, and a subsequent centripetal cell migration during which phenotypic transition from glomerulosa into fasciculata and reticularis cells and elimination of senescent cells through apoptosis occur. As the molecular mechanisms implied in adrenocortical cell migration are still generally unknown, we addressed that question in the present study. Of several extracellular matrix proteins tested, laminin was the most potent chemotactic and haptotactic factor for bovine fasciculata adrenocortical cells. The maximal chemotactic effect (3-fold stimulation) was observed with 50-75 micrograms/ml laminin, whereas the haptotactic effect (3.5-fold stimulation) plateaued for laminin concentrations in the coating solution over 25 micrograms/ml. Using an anti-Engelbreth-Holm-Swarm laminin antibody, we could demonstrate that adrenocortical cells actively synthesize and secrete Engelbreth-Holm-Swarm-laminin, with the A chain produced in limiting quantities. ACTH treatment of adrenocortical cells specifically induced a 2.7- to 4.5-fold increase in A chain synthesis, resulting in a corresponding increase in the amount of secreted laminin. The distribution of laminin in the adrenal cortex tissue was then evaluated by standard immunohistochemistry. The protein appeared to be uniformly expressed in the three zones of the cortex. This observation does not favor the hypothesis that laminin acts as an attractant driving centripetal cell migration. Laminin, which is synthesized under the control of the systemic hormone ACTH, appears as a permissive factor that facilitates proper homeostasis of the adrenocortical tissue.

Adrenal Cortex↗
PubMed1996

Opposite regulation of thrombospondin-1 and corticotropin-induced secreted protein/thrombospondin-2 expression by adrenocorticotropic hormone in adrenocortical cells.

Corticotropin-induced secreted protein (CISP) is a trimeric glycoprotein secreted by primary cultures of bovine adrenortical cells in response to adrenocorticotropic hormone (ACTH). This protein was recently purified in our laboratory, and its N-terminal amino-acid sequence revealed a significant similarity with thrombospondin-2 (TSP2). We report here the nucleotide sequence of a 386 bp RT-PCR fragment specific for CISP. The deduced protein sequence shares 84% identity with the N-terminal portion of mature human TSP2, suggesting that CISP is its bovine counterpart. Northern analysis of adrenocortical cell RNA using the above cDNA fragment as a probe revealed a 6.0 kb CISP/TSP2 mRNA whose abundance was increased nearly fivefold following a 24 h cell treatment with 10(-7) M ACTH. Under the same conditions, the expression of TSP1 mRNA was reduced by tenfold. The protein levels of TSP1 and CISP/TSP2 varied accordingly with their respective mRNA levels, as shown by immunoprecipitation and immunofluorescence experiments. Taken together, these data show that ACTH induces a dramatic shift in the pattern of adrenocortical cell thrombospondin expression from TSP1 to CISP/TSP2. This observation suggests that these two members of the thrombospondin family exert distinct biological functions in the adrenal cortex. This hypothesis is further supported by the observation that anti-CISP antibodies inhibit the maintenance of the morphological changes of bovine adrenocortical cells induced by ACTH, whereas anti-TSP1 antibodies do not.

Adrenal Glands↗
PubMed1996

ACTH angiotensin II and TGF beta participate in the regulation of steroidogenesis in bovine adrenal glomerulosa cells.

Bovine zona glomerulosa cells, on the first day of culture, produce aldosterone as their major steroid with no detectable cortisol secretion. Continuous incubation with ACTH had no effect on aldosterone production nor on aldosterone synthase activity. This treatment resulted in a dose and time dependent rise in 17 alpha-hydroxylase activity, in parallel with an increase in cytochrome P-450(17 alpha) (CYP17) protein and mRNA. We have previously shown that TGF beta 1 is a potent inhibitor of differentiated functions of bovine fasciculata-reticularis cells and that CYP17 and AII receptors are the major targets explaining this effect. The present study examined whether 17 alpha-hydroxylase activity in glomerulosa cells could be regulated by angiotensin II (AII) and transforming growth factor-beta 1 (TGF beta 1). AII inhibits the induction of CYP17 by ACTH in a dose dependent manner. TGF beta 1 also blocks almost completely the stimulatory effect of ACTH. In order to suppress the endogenous action of TGF beta 1, incubations were performed with an anti-TGF beta antibody. This specific antibody induces the expression of CYP17 resulting in increased activity and mRNA levels. These results show that AII is able to modulate the expression of CYP17 in adrenal glomerulosa cells following ACTH stimulation. Furthermore, TGF beta 1 exerts an autocrine effect on the differentiation of glomerulosa cells through a regulatory loop repressing CYP17 activity.

Adrenocorticotropic Hormone↗
PubMed1996

Alpha 2-macroglobulin: a binding protein for transforming growth factor-beta and various cytokines.

alpha 2-Macroglobulin (alpha 2M) is a large plasma glycoprotein that has long been known as an irreversible inhibitor of a variety of proteinases. More recently, it has been reported that numerous growth factors, cytokines and hormones bind to alpha 2M through diverse mechanisms. We review here a series of observations from our laboratory that support the concept that alpha 2M is a carrier protein for transforming growth factor-beta (TGF-beta) and allows this factor to act as an autocrine regulator of adrenocortical steroidogenic functions. alpha 2M was found to by synthesized and secreted by primary cultures of bovine adrenocortical cells in fairly large amounts (1 microgram/10(6) cells/24 h). TGF-beta is also secreted by this cell type, although under a latent form. Two distinct latent TGF-beta complexes have been characterized in adrenocortical cell conditioned medium, one of which is a complex between alpha 2M and TGF-beta. Although alpha 2M prevents the binding of TGF-beta to its membrane receptors, long-term incubation of alpha 2M with adrenocortical cells results in inhibition of cortisol production similar to that observed in the presence of TGF-beta alone. Taken together, these observations suggest that adrenocortical cells can release active TGF-beta from its latent complex with alpha 2M through an unknown mechanism. alpha 2M can therefore be considered as a TGF-beta carrier protein.

Adrenal Cortex↗
PubMed1994

Synergistic induction of alpha 2-macroglobulin synthesis by fibroblast growth factor-2 and transforming growth factor beta 1 in bovine adrenocortical cells.

We report here that basic fibroblast growth factor (FGF-2), a potent mitogen for adrenocortical cells, stimulates the expression of alpha 2-macroglobulin by these cells at a transcriptional level and is synergistic with TGF beta 1 for this effect. This is supported by the following observations: (i) Treatment of adrenocortical cells by FGF-2 resulted in a time-dependent and dose-dependent increase of alpha 2M synthesis, (ii) FGF-2 did not modify alpha 2M secretion rate; (iii) The induction of alpha 2M synthesis by FGF-2 was not observed in the presence of the transcription inhibitor DRB; (iv) The amount of alpha 2M mRNA was increased by 2 to 3 fold under either FGF-2 or TGF beta 1 treatment; (v) Optimal doses of TGF beta and FGF-2 synergistically increased alpha 2M synthesis. Since alpha 2M is a growth factor-binding protein, its regulation by FGF-2 may represent an important feedback mechanism controlling the bioactivity of autocrine regulators (FGF-2, TFG beta) of adrenocortical functions.

Animals↗
PubMed1992

Inhibition of adrenocortical steroidogenesis by alpha 2-macroglobulin is caused by associated transforming growth factor beta.

alpha 2-Macroglobulin (alpha 2M) is the major protein secreted by bovine adrenocortical cells in primary culture and its synthesis is stimulated by transforming growth factor beta (TGF beta). We investigated here the effects of alpha 2M on adrenocortical steroidogenesis. We observed that commercial preparations of bovine plasma alpha 2M were able to mimic the inhibitory action of TGF beta on adrenocortical cortisol production, with the same specificity of action directed at the steroid 17 alpha-hydroxylation step. This inhibition was time-dependent and dose-dependent (50% inhibition observed with 2 mg/ml alpha 2M). Acid/ethanol extracts of alpha 2M appeared to retain the full inhibitory activity of alpha 2M. Anti-TGF beta antibodies could reverse the inhibition caused by the acid/ethanol extract but not that caused by native alpha 2M. Taken together, these results indicate that the inhibition of adrenocortical steroidogenesis induced by alpha 2M is caused by associated TGF beta. We estimated that 2 mg of alpha 2M contained approximately 0.1 ng of TGF beta, corresponding to a molar ratio of 1/700,000 between TGF beta and alpha 2M. These results also clearly indicate that the alpha 2M-TGF beta complexes are biologically active on adrenocortical cells, suggesting that these cells possess the enzymatic equipment that can activate the latent alpha 2M-TGF beta complexes.

Adrenal Cortex↗
PubMed1991

Production of platelet-activating factor is a component of the angiotensin II-protein kinase C activation pathway in bovine adrenocortical cells.

Lyso-platelet-activating factor (lyso-PAF): acetyl-CoA acetyltransferase (EC 2.3.1.67) enzyme activity was characterized for the first time in bovine adrenocortical tissue. It was found to be associated with the microsomal membrane fraction, in which it exhibited a specific activity of 0.4 nmol/min per mg of protein and catalytic properties similar to those described in other cell types. The adrenocortical acetyltransferase activity was increased by 2-3-fold on incubation of the preparation with purified protein kinase C (PKC) under phosphorylating condition. This activation was optimal after 5 min of incubation and paralleled an increase in PKC-catalysed 32P incorporation into microsomal proteins. Both acetyltransferase activation and protein phosphorylation were dependent on the presence of Ca2+ and phospholipids, and were blocked in the presence of the potent PKC inhibitor H-7. In the intact adrenocortical cell, angiotensin II and a potent phorbol ester (phorbol 12-myristate 13-acetate) were able to rapidly induce an increase in the biosynthesis of PAF, which was mostly released into the extracellular medium. These data suggest that bovine adrenocortical lyso-PAF acetyltransferase may be regulated by a PKC-dependent activation pathway, whereas no evidence for an additional adrenocorticotropin/cyclic AMP-dependent stimulation process was obtained in this cell type. Bovine adrenocortical cell membrane preparations were shown to possess high-affinity PAF-binding sites (Kd approximately 0.5 nM). Altogether, these observations suggest that PAF production and release may play a role in the autocrine or paracrine control of adrenocortical cell activation.

Acetyltransferases↗
PubMed1991

Transforming growth factor beta 1: an autocrine regulator of adrenocortical steroidogenesis.

Transforming growth factor beta 1 (TGF beta 1) is a member of a large family of structurally related regulatory polypeptides which comprises both functionally similar (TGF beta 1, TGF beta 2, TGF beta 3, TGF beta 4 and TGF beta 5) and functionally distinct proteins. In the past few years, TGF beta 1 has emerged as a multifunctional protein. One of its remarkable properties is its capacity to negatively modulate the differentiated, steroidogenic adrenocortical functions. We present here a review of the results from our recent work related to the effects of TGF beta 1 on bovine adrenocortical cell (zona fasciculata-reticularis) functions. We identified the steroid 17 alpha-hydroxylase (P-450 17 alpha) biosynthetic enzyme and the angiotensin II receptor as major targets whose expression are negatively regulated by TGF beta 1 in these cells. We characterized TGF beta 1 receptors at the surface of adrenocortical cells (mainly type I and type III receptors) and observed that their number is increased under ACTH treatment. Furthermore, we could detect the presence of immunoreactive TGF beta 1 in the bovine adrenal cortex whereas it was undetectable in the adrenal medulla and in the capsule. We also observed that adrenocortical cells secrete TGF beta 1 under a latent form together with large amounts of alpha 2-macroglobulin, a protease inhibitor known to be implied in the latency of TGF beta in serum. Taken together, these observations led us to a working hypothesis, proposing TGF beta 1 as an autocrine and/or paracrine regulator of adrenocortical steroidogenic functions. This concept points out the physiological activation of the latent TGF beta 1 complex as the important limiting step controlling its action in the adrenal cortex.

Adrenal Cortex↗
PubMed1991

Direct regulating effects of transforming growth factor-beta 1 on lactate production in cultured porcine Sertoli cells.

In the present study we have tested the direct effects of transforming growth factor-beta 1 (TGF beta 1) on lactate production by Sertoli cells isolated from immature porcine testes. In Sertoli cells cultured in a defined medium, TGF beta 1 was shown to stimulate lactate production in a time- and dose-dependent manner. The maximal and half-maximal effects of TGF beta 1 on lactate production were obtained in the picomolar concentration range, respectively 24 and 8 pM TGF beta 1. TGF beta 1 action was found closely related to that of insulin since 1) both TGF beta 1 (40 pM) and insulin (1 microgram/ml) induced the secretion of similar and nonadditive amounts of lactate; and 2) TGF beta 1 and insulin induced comparable increases in lactate production in FSH (1 microgram/ml)-treated Sertoli cells. Because lactate is derived from glucose, 2-deoxy-D-glucose (2-DOG) was used to investigate the hexose transport system of Sertoli cells after insulin, FSH, and TGF beta 1 treatments. Insulin (1 microgram/ml) and FSH (1 microgram/ml) were found to stimulate 2-DOG transport with a similar time course, with an effect detected up to 30 min and maximal at 150 min. In contrast, although TGF beta 1 also enhanced 2-DOG uptake by Sertoli cells, the increase in glucose transport was delayed, since the TGF beta 1 effect was first detected at 150 min and was maximal at 360 min. These effects of TGF beta 1 action on Sertoli cell activity are exerted through specific membrane TGF beta 1 receptors. Scatchard analysis of the binding of TGF beta 1 to cultured Sertoli cells revealed the presence of both a high affinity (Kd, approximately 180 pM) and a low affinity binding site systems for TGF beta 1. Affinity labeling of these receptors by covalent attachment to [125I] TGF beta 1 with disuccinimidyl suberate and subsequent electrophoretic analysis of the labeled complexes revealed the specific binding of [125I] TGF beta 1 to three predominant molecules of 260, 130, and 70 kDa. In conclusion, the present study demonstrates that testicular Sertoli cells are targets for TGF beta 1 action. In view of the importance of lactate as a substrate for germ cells, it is suggested that TGF beta 1 might also be involved in the development of normal germinal epithelium.

Animals↗
PubMed1991

Immunolocalization of transforming growth factor-beta 1 in the bovine adrenal cortex using antipeptide antibodies.

Eight- to 12-amino acid long peptides, representing fragments of transforming growth factor-beta 1 (TGF beta 1) and TGF beta 2 were selected on the basis of their potential immunogenicity and were used to generate polyclonal antibodies. Anti-TGF beta 1-(91-103) antibodies recognized specifically TGF beta 1, prevented TGF beta 1 binding to NRK-49F cells, and neutralized the biological activity of TGF beta 1 in adrenocortical cells (consisting in the inhibition of angiotensin-II-induced cortisol production). Antibodies raised against TGF beta 2-(65-73) appeared to recognize TGF beta 2 with a better affinity than TGF beta 1, but were unable to block the binding of either TGF beta 1 or TGF beta 2 to their receptors or to inhibit their biological activity. These observations are in line with a prominant role of the C-terminal domain of TGF beta 1 in its interaction with its receptor(s) and, hence, in its biological activity. Using anti-TGF beta 1-(91-103) antibodies, we could localize immunoreactive TGF beta 1-like material in the cortex of adult bovine adrenal glands. No reactivity was detected in the capsule or adrenal medulla. The reactivity was maximal in the zona fasciculata/reticularis and weaker in the zona glomerulosa. TGF beta-like material was present in a latent form in the conditioned medium from primary cultures of bovine adrenocortical cells. These cells secreted about 5 ng heat-activatable TGF beta-like material/24 h of culture.10(6) cells. Taken together with our previous reports that bovine adrenocortical cells possess high affinity TGF beta 1 receptors and secrete a TGF beta 1-like molecule under a latent form, the present observations further support the hypothesis that TGF beta 1 or a closely immunologically related protein acts as an autocrine regulator of adrenocortical steroidogenic functions.

Adrenal Cortex↗