Crinopexy: extracellular regulation of growth factor action.
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Animals↗
Biomedical subjects
J J Feige
Publications and source records attributed to J J Feige.
Alpha 2-macroglobulin and the control of adrenocortical steroidogenic function.
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Adrenal Cortex↗
Expression of fibroblast growth factor receptor-2 splice variants is developmentally and tissue-specifically regulated in the amphibian embryo.
There is increasing evidence that distinct fibroblast growth factor receptor (FGFR) genes are involved in embryonic development and that unique expression patterns of individual FGFRs correlate with tissue-specific functions. In addition, alternative splicing of mRNA transcripts from at least two of these genes (FGFR-1 and FGFR-2) can generate receptor variants with different ligand-binding specificity. By polymerase chain reaction methods and by screening a cDNA library, we have isolated five amphibian FGFR-2 splice variants which share a high degree of identity to their human counterparts. These mRNAs are developmentally regulated and are expressed in a tissue-specific manner. In particular, two alternative exons (termed IIIb and IIIc) in the second half of the third immunoglobulin-like loop are remarkably conserved and have a distinct pattern of regulation during development. Either aFGF or bFGF can activate IIIb- or IIIc-containing receptors. In contrast, KGF only activates IIIb-containing receptors. Exon IIIb-containing receptors are maternally derived mRNAs, whereas exon IIIc-containing receptors are zygotically expressed. Furthermore, their tissue distribution pattern was mutually exclusive. From the beginning of the neurula stage onward, IIIb transcripts are expressed in the epidermis, while IIIc transcripts are activated in the neuroectoderm by neural induction. At the late tail-bud stage, in situ hybridization revealed expression of IIIc mRNA in the telencephalon and the diencephalon, as well as in the head mesenchyme condensation sites originating from the proliferating neural crest cells. IIIb mRNA was detected in the epidermis and in the epithelium of the pharynx. Our data suggest that exon IIIb-containing receptors may play a role in the development of epithelial tissues, while exon IIIc-containing receptors may play a role during neural tissue formation.
Amino Acid Sequence↗
Steroidogenic adrenocortical cells synthesize alpha 2-macroglobulin in vitro, not in vivo.
We previously identified alpha 2-macroglobulin as the major protein secreted by primary cultures of adrenocortical cells. We report here that in the adrenal gland, the distribution of alpha 2-macroglobulin in the adrenocortical tissue is restricted to the endothelium of blood vessels and that no immunoreactivity is found in steroidogenic cells. A time course study revealed that freshly dissociated bovine adrenocortical cells were void of alpha 2-macroglobulin immunoreactivity whereas the proportion of alpha 2-macroglobulin-positive cells reached more than two-thirds of the population between day 4 and day 7 of culture. Double immunoenzymatic labeling of 6-day-old cultures revealed a co-localization of alpha 2-macroglobulin and the steroidogenic enzyme P-450SCC. Treatment of 5-day-old cultures (expressing alpha 2-macroglobulin) for 24 h by either ACTH (10(-9)-10(-6) M) or alpha 2-macroglobulin (2.5 mg/ml) resulted in a marked decrease of the expression of alpha 2-macroglobulin. These data indicate that ACTH and plasmatic alpha 2-macroglobulin could physiologically repress alpha 2-macroglobulin expression in the adrenal cortex in vivo.
Adrenal Cortex↗
Distinct effects of thrombospondin-1 and CISP/thrombospondin-2 on adrenocortical cell spreading.
Corticotropin-induced secreted protein (CISP) is a trimeric protein secreted by bovine adrenocortical cells in response to ACTH, that is likely to represent the bovine form of thrombospondin-2 (TSP2). This study was aimed at delineating the respective effects of CISP/TSP2 and TSP1 (thrombospondin-1) on adrenocortical cell attachment and spreading. TSP1 and CISP/TSP2 were found to slightly reduce the attachment of adrenocortical cells to plastic in the presence of serum but exhibited a pronounced differential effect on cell spreading. CISP/TSP2 inhibited adrenocortical cell spreading in a dose-dependent manner (maximal effect with 40 micrograms/ml) whereas TSP1 (up to 100 micrograms/ml) did not influence this process. The inhibition of spreading was observed whether plates were coated with CISP/TSP2 alone or with a mixture of CISP/TSP2 and fibronectin. We suggest that the inhibition of in vitro adrenocortical cell spreading by CISP/TSP2 is indicative of an implication of this protein in the migration of adrenocortical cells in vivo.
Adrenal Cortex↗
Growth factors and pericellular components as a local signalling relay in the action of systemic hormones.
Recent years have shown that peptidic signals, originally identified as growth factors, may represent a local signalling system involved in the regulation of cell function under the dependence of systemic hormones. As an example, (i) adrenocortical cells are highly sensitive to transforming growth factor-beta (TGF beta) which is a powerful inhibitor of their differentiated functions; (ii) adrenocortical cells produce and secrete TGF beta, which is present in the adrenal cortex in situ; (iii) adrenocorticotropin (ACTH), which is the best known positive effector of adrenocortical functions induces an up-regulation of TGF beta receptors in adrenocortical cells. Altogether, these observations suggest that TGF beta may be a local signalling system acting in balance with the systemic hormone ACTH to eventually tune the ability of adrenocortical cells to produce corticosteroids.
Adrenal Cortex↗
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↗
[Signal transmission modalities induced by growth factors: from membrane to nucleus].
Growth factors transduce their biological signals through transmembrane receptors that possess an intrinsic or an associated protein-tyrosine kinase activity. Two complete signalling pathways, from membrane to nucleus, were elucidated in 1993. They are described in detail in this review.
Calcium-Calmodulin-Dependent Protein Kinases↗
The molecular structure of corticotropin-induced secreted protein, a novel member of the thrombospondin family.
CISP (corticotropin-induced secreted protein) is a secreted protein recently purified in our laboratory from the conditioned medium of ACTH-treated bovine adrenocortical cells. Partial amino acid sequencing of CISP revealed homology with thrombospondins (TSPs), a family of adhesive proteins and in particular with TSP2. We report here the characterization of the molecular structure of CISP. Analysis of CISP by polyacrylamide gel electrophoresis in the absence or presence of SDS indicated an apparent molecular mass approximately equal to 600 kDa for the unreduced protein and an apparent molecular mass of 195 kDa after reduction by 2-mercaptoethanol. The sedimentation coefficient of CISP determined by ultracentrifugation on sucrose gradients was shifted from 9.7 S in the absence to 5.7 S in the presence of 2-mercaptoethanol. These data are consistent with a trimeric organization of the CISP molecule in which 195-kDa monomers would be linked together by disulfide bonds. The trimeric structure of CISP could be observed by rotary shadowing/electron microscopy, where CISP appeared to be composed of three equally electron-dense nodules and of a fourth nodule formed by the close association of three smaller fragments. The overall size of the molecule was 60 nm. We also observed that CISP is sulfated and glycosylated. Using glycosylation inhibitors, we could determine that CISP is synthesized as a 175-kDa core protein, is then matured into a 190-kDa high-mannose form and secreted as a 195-kDa mature protein. Inhibition of sulfation by chlorate did not prevent CISP secretion, whereas inhibition of glycosylation by tunicamycin blocked it. Taken together, these data indicate that the TSP2-related CISP molecule presents both structural and functional properties very similar to those of TSP1. CISP differs greatly, however, from TSP1 by the inducibility of its synthesis by cAMP.
Adrenal Cortex↗
Corticotropin-induced secreted protein, an ACTH-induced protein secreted by adrenocortical cells, is structurally related to thrombospondins.
The treatment of primary cultures of bovine adrenocortical cells with nanomolar concentrations of ACTH induces a 10-fold increase in the synthesis of a secreted protein of apparent molecular mass 195 kDa on reducing SDS-polyacrylamide gels. This corticotropin-induced secreted protein (CISP) appears to be an oligomeric calcium-binding protein. Its secretion under serum-free culture conditions is sustained over 4 days in the continuous presence of ACTH. Induction of CISP secretion by ACTH is mimicked by cAMP analogs and adenylate cyclase activators. We report here the purification of CISP to apparent homogeneity with an overall yield of 43% using a combination of heparin-agarose and Mono-Q chromatographies. The NH2-terminal amino acid sequence and the sequence of several tryptic peptides revealed that CISP is structurally related to the members of the thrombospondin (TSP) family. Among these members, bovine CISP appeared to be more homologous to mouse TSP2 (85% identity in the 29 amino acid long NH2-terminal sequence) than to TSP1 (18% identity in the same region). We also observed that CISP binds Ca2+ and is an adhesive protein for bovine adrenocortical cells. Thus, CISP possesses both structural and functional properties of thrombospondins. Whether CISP represents the bovine form of TSP2 or a novel member of the expanding thrombospondin family will need to be elucidated by cloning and sequencing of a larger portion of the molecule.
Adrenal Cortex↗
Transforming growth factor beta 1 and adrenocorticotropin differentially regulate the synthesis of adrenocortical cell heparan sulfate proteoglycans and their binding of basic fibroblast growth factor.
Adrenocortical differentiated functions are under the control of both endocrine hormones such as ACTH and local factors such as transforming growth factor beta (TGF beta) or basic fibroblast growth factor (bFGF). Besides their regulatory actions on the synthesis of corticosteroids, these two classes of factors also exert some important effects on the cellular environment. We have examined here the regulation by ACTH and TGF beta of adrenocortical cell proteoglycan synthesis and secretion. Under basal conditions, adrenocortical cells synthesized and secreted several species of sulfated proteoglycans, 80% of them being recovered in solution in the culture medium. When analyzed by ion exchange chromatography, the cell extracts and the media from cells metabolically labeled with 35S-sulfate were found to contain two and three species of radioactive sulfated proteoglycans, respectively. All species were proteoheparan-sulfates. Treatment of adrenocortical cells with TGF beta 1 or ACTH resulted in a significant increase of the incorporation of 35S into both secreted and cell-associated proteoglycans. ACTH stimulated more than three times the amount of secreted proteoglycans eluting from DEAE-Trisacryl as peak B, whereas TGF beta preferentially increased the amount of peak C. No important modification of the size of the synthesized proteoglycans was observed. The subpopulation of heparan sulfate proteoglycans capable to bind bFGF was also largely increased after ACTH or TGF beta treatment and paralleled the variation in overall proteoheparan sulfate synthesis. Thus those effects of TGF beta and ACTH on proteoglycan synthesis may participate in an increased ability of adrenocortical cells to bind and respond to bFGF.
Adrenal Cortex↗
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↗
Basic fibroblast growth factor enhances testosterone secretion in cultured porcine Leydig cells: site(s) of action.
The effect and mechanism of action of basic fibroblast growth factor (bFGF) on testicular steroidogenesis were investigated using as a model primary cultures of purified porcine Leydig cells from immature intact animals. Basic FGF increased basal and human chorionic gonadotrophin (hCG)-induced testosterone accumulation (with an ED50 of 0.64 ng/ml bFGF, 35 pM) in the medium following a long-term treatment. The effects of bFGF (10 ng/ml, 72 h) were found at all hCG concentrations tested (0.001-1 ng/ml), the growth factor affecting the maximal steroidogenic capacity of the Leydig cells but not their sensitivity to the gonadotrophin. In this context, we have therefore investigated whether the stimulatory effect of bFGF on testosterone formation was related to an increase of the steroidogenic enzyme activities. The data obtained indicate that the growth factor did not affect the gonadotrophin action on the formation of delta 5-steroid hormone, namely dehydroepiandrosterone (DHEA) (evaluated in the presence of 10(-5) M WIN 24540, an inhibitor of 3 beta-hydroxysteroid dehydrogenase/isomerase). By contrast, bFGF (10 ng/ml, 72 h) was found to increase in a comparable manner the conversion of pregnenolone, DHEA and delta 4-androstenedione into testosterone, suggesting a stimulatory effect on 17 beta-hydroxysteroid dehydrogenase activity. Indeed, bFGF enhanced in a dose-dependent manner (ED50 = 39 pM) this enzyme activity evaluated through the conversion of delta 4-androstenedione to testosterone. These effects of bFGF on Leydig cell steroidogenic activity are probably exerted through specific membrane bFGF receptors.(ABSTRACT TRUNCATED AT 250 WORDS)
17-Hydroxysteroid Dehydrogenases↗
Differential expression and regulation of two distinct fibroblast growth factor receptors during early development of the urodele amphibian Pleurodeles waltl.
Fibroblast growth factor (FGF) has been shown to be involved in mesoderm induction during amphibian development. Its presence in the embryo suggests that FGF is an endogenous inducer. By polymerase chain reaction (PCR) methods and by screening a Pleurodeles waltl tail-bud cDNA library with a cDNA probe for human FGF receptor, we have isolated two full-length cDNA clones, which we designate PFR1 and PFR4 based on their homology to the human FGF receptors FGFR-1 and FGFR-4. Both cDNA clones encode Pleurodeles FGF receptors that share characteristics common to members of the FGF receptor superfamily. The deduced amino acid sequence of PFR1 is 85% identical overall with the human fms-like-gene (FLG). PFR4 is most closely related to the human FGFR-4 (66% overall identity). The tyrosine kinase catalytic domains of both receptors are remarkably conserved. The two receptors show distinct patterns of regulation during early development. PFR1 first appears as a maternally derived mRNA and mRNA levels remain constant during early developmental stages. However, PFR4 mRNA is first expressed at the late blastula stage, which suggests that its expression is a result of zygotic transcription. Furthermore, northern blot analysis indicates that PFR1 mRNA is distributed evenly in the early gastrula while PFR4 mRNA is predominantly localized to the presumptive ectoderm. At tail-bud stage, PFR1 transcripts are localized primarily to the neural and mesodermal tissues, PFR4 transcripts are most abundantly expressed in neural tissue, and more transcripts are detected in lateral plate mesoderm than in the somites. When animal cap explants of blastulae are cultured in the presence of mesoderm-inducing factors, PFR1 mRNA levels are maintained by bFGF and activin A. In contrast, PFR4 mRNA levels are significantly down-regulated. These observations suggest a differential expression and regulation of FGF receptors in early amphibian development.
Amino Acid Sequence↗
Phosphorylation of basic fibroblast growth factor by purified protein kinase C and the identification of a cryptic site of phosphorylation.
We have further characterized the protein kinase C (PK-C) dependent phosphorylation of basic fibroblast growth factor (FGF). Intact recombinant basic FGF and a series of ten peptide fragments of basic FGF were phosphorylated by PK-C and the products were analyzed by SDS-PAGE and autoradiography. As expected, peptide fragments containing the known site of phosphorylation (Ser64) are substrates for phosphorylation. Surprisingly however, peptides containing the receptor binding domain of the mitogen [basic FGF(106-115)] are also phosphorylated. An examination of this sequence reveals the presence of a consensus sequence (Ser108-Ala109-Lys110) that mediates the reaction. Accordingly, all peptides that contain the core amino acids basic FGF(106-111) are substrates for phosphorylation. Peptide mapping of basic FGF confirms that Ser64 is the primary site of phosphorylation, suggesting that Ser108 is a cryptic consensus sequence. Because basic FGF is metabolized to sequence specific fragments after its binding and internalization into target cells, this cryptic site may in fact be phosphorylated in vivo.
Animals↗
Phosphorylation and identification of phosphorylated forms of basic fibroblast growth factor.
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Adenosine Triphosphate↗
Growth factor regulation of adrenal cortex growth and function.
The control of adrenal cortex growth in vivo during development or under certain stress conditions is still very poorly understood at the molecular level. Some information can be collected however from in vitro experiments. Acidic and basic FGF appear to be the most potent mitogens, so far, for primary cultures of adult adrenocortical cells, whereas EGF can also stimulate growth of fetal cells. Several growth factors have emerged in the recent years as multifunctional molecules that play important regulatory functions on adrenocortical steroidogenesis. These include EGF, IL-1, insulin, IGF-1 and TGF beta. In certain cases (e.g. IGF-1, TGF beta), these factors participate in autocrine loops of regulation. The differential expression, release and activation of these factors might locally regulate the steroidogenic action of the hormonal signals delivered through the hypothalamo-pituitary-adrenal axis.
Adrenal Cortex↗
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↗