cAMP-mediated regulation of adrenocortical cell bFGF receptors.
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Adrenal Cortex↗
Biomedical subjects
J J Feige
Publications and source records attributed to J J Feige.
Transforming growth factor beta 1 is a negative regulator of steroid 17 alpha-hydroxylase expression in bovine adrenocortical cells.
Transforming growth factor beta 1 (TGF-beta 1) has been shown previously to induce striking alterations of bovine adrenocortical cell steroidogenic functions. One of the major lesions was characterized as a loss of steroid-17 alpha-hydroxylase activity, a key step in the biosynthetic pathway leading to active corticosteroid hormones. The mechanism of this negative effect of TGF-beta 1 on adrenocortical differentiated functions was investigated. It was observed that: 1) bovine adrenocortical 17 alpha-hydroxylase activity rapidly decreased in cells exposed to TGF-beta 1, in a time (10-20 h)-dependent manner; 2) immunoblotting of the corresponding cytochrome P-450(17) alpha showed that the loss of activity was superimposable to the decrease of the cellular protein content; 3) the cell content in 17 alpha-hydroxylase messenger RNA sharply dropped under TGF-beta 1 treatment (70-75% loss within 3-4 h) as determined by Northern blot analysis; 4) TGF-beta 1 inhibited as well the induction of P-450(17) alpha normally observed under adrenocorticotropin treatment; and 5) these TGF-beta 1 effects were selectively directed toward P-450(17) alpha expression, whereas another major steroidogenic cytochrome, i.e. P-450scc, was not affected. These observations showed that TGF-beta 1 is a potent negative modulator of 17 alpha-hydroxylase expression in bovine adrenocortical cells, very possibly at the transcriptional level. TGF-beta 1 (whose gene is expressed in these cells) may thus be examined as a possible autocrine inhibitory factor implied in the regulation of adrenocortical differentiated functions, in balance with ACTH, which represents the major positive signal in this system.
Adrenal Cortex↗
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↗
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↗
Proteoheparan sulfates contribute to the binding of basic FGF to its high affinity receptors on bovine adrenocortical cells.
Bovine adrenocortical cells in primary culture express the basic fibroblast growth factor (bFGF) gene and their proliferation is stimulated by this growth factor. We report here the characterization of bFGF receptors on these cells. Binding studies revealed the presence of two bFGF receptor types: a limited number (4,300 sites/cell) of high affinity sites (Kd congruent to 2 pM) and a larger number (230,000 sites/cell) of lower affinity sites (Kd congruent to 400 pM). Cross-linking of 125I-bFGF to adrenocortical cells revealed two bands at 145 kDa and 125 kDa which are attributed to molecular complexes between the high affinity receptors and their ligand. These high affinity receptors possess N-linked carbohydrate chains that are important for proper cell surface expression but are devoid of glycosaminoglycan chains. The low-affinity (2 M NaCl-sensitive) binding sites are totally degraded by heparitinase treatment of adrenocortical cells indicating that low-affinity sites are borne by heparan sulfate proteoglycans. However, heparitinase treatment also reduced partially the binding of bFGF to high-affinity (2 M NaCl-resistant) sites. This argues for a contribution of heparan sulfate proteoglycans to the binding of bFGF to high-affinity receptors. Exogenous soluble heparin or heparan sulfate did not restore normal high affinity bFGF binding onto heparitinase-treated cells, suggesting that heparan sulfate proteoglycans either must be membrane-anchored or must contain specific structural features to enhance FGF binding to high-affinity receptors. Taken together with previous reports, this work supports the hypothesis that bFGF may act as an autocrine growth factor in the adrenal cortex.
Adrenal Cortex↗
[Autocrine regulation of adrenal steroidogenesis by growth factor transforming TGF-beta 1].
Transforming growth factor beta (TGF beta) is a multifunctional polypeptide presenting various biological activities depending on the cellular context where it is studied. In the adrenal cortex, TGF beta is a potent inhibitor of corticosteroïdogenesis. The sum of data that we have collected in the past years has allowed us to establish that TGF beta (in particular TGF beta 1) is implicated in a physiological autocrine/paracrine regulatory loop. We determined that primary cultures of bovine fasciculatareticularis cells synthesize and secrete about 5 ng/24 h x 10(6) cells of TGF beta but that most of this activity is under a latent form. In the adrenal gland, TFG beta 1 could be localized in the fasciculata-reticularis zone by immunohistochemistry. We also reported that adrenocortical cells possess type I and type III (betaglycan) receptors, whose number is positively regulated by ACTH. Moreover, we have identified 3 major targets of TGF beta in our cell model: cytochrome P-450(17) alpha (17 alpha-hydroxylase) whose gene expression is decreased, angiotensin II receptors and LDL receptors whose number is reduced after TGF beta treatment. As a conclusion, TGF beta appears to participate together with other regulatory peptides (IGF-1, TNF alpha) in the fine tuning of corticosteroid secretion levels which are under the major control of the hypothalamo-pituitary axis.
Adrenal Cortex↗
Transforming growth factor-beta stimulates the expression of alpha 2-macroglobulin by cultured bovine adrenocortical cells.
Adrenocortical cell major secreted protein was purified from the conditioned medium of primary cultures of bovine adrenocortical (BAC) cells. Immunochemical analysis and N-terminal sequencing of the purified protein identified it to alpha 2-macroglobulin (alpha 2-M). It appeared that 15 out of the 17 N-terminal amino acids were conserved between adrenocortical cell major secreted protein and human alpha 2-M. Study of alpha 2-M production by BAC cells revealed that its secretion was stimulated severalfold by transforming growth factor-beta 1 (TGF-beta 1). The stimulation occurred in a time-dependent (reaching a plateau at 24 h) and dose-dependent (ED50 = 0.1 ng/ml TGF-beta 1) manner. It was blocked when BAC cells were exposed to 5,6-dichlorobenzimidazole riboside, a potent inhibitor of RNA polymerase II, suggesting that TGF-beta 1 acts as an activator of alpha 2-M gene expression at the transcriptional level. Northern blot analysis confirmed that the alpha 2-M mRNA level was increased (4-fold) in BAC cells following TGF-beta 1 treatment. TGF-beta 2, TGF-beta 1,2, basic fibroblast growth factor, and angiotensin II also appeared able to stimulate alpha 2-M secretion in BAC cells, whereas adrenocorticotropin was strongly inhibitory. Given the previous reports that TGF-beta 1 is a potent inhibitor of adrenocortical steroidogenesis (Feige J.J., Cochet, C., Rainey, W.E., Madani, C., and Chambaz, E. M. (1987) J. Biol. Chem. 262, 13491-13495) and that alpha 2-M is a TGF-beta 1-binding protein, these observations suggest that alpha 2-M may play an important role in conjunction with hormones and growth factors in the homeostatic regulation of adrenocortical functions.
Adrenal Cortex↗
Stimulation of fibronectin production by TGF-beta 1 is independent of effects on cell proliferation: the example of bovine adrenocortical cells.
We reported previously that transforming growth factor beta (TGF-beta) does not influence the proliferation of bovine adrenocortical cells but is a very potent inhibitor of their steroidogenic functions (Feige et al.: Journal of Biological Chemistry 262:13491-13495, 1987). In the present study, we addressed the question of whether these cells modify the synthesis of their extracellular matrix (in particular of fibronectin) in response to TGF-beta 1, similarly to the changes observed in cell types whose growth is modified by this factor (e.g., fibroblasts). Immunofluorescence studies using anti-fibronectin antibodies revealed that TGF-beta 1 treatment in serum-free medium induced the formation of fibronectin-containing fibrils associated with adrenocortical cells. Metabolic labeling of adrenocortical cells with [35S]-methionine showed that fibronectin synthesis and secretion were highly stimulated by low concentrations of TGF-beta 1. Half-maximal stimulation was observed for TGF-beta 1 concentrations in the range of 0.1 to 0.5 ng/ml and maximal stimulation reached 35-fold over control at the concentration of 2 ng/ml. The earlier detectable effect was observed after 8 h of treatment (6-fold stimulation) and the maximal increase was reached after 24 h of treatment. Stimulation of adrenocortical fibronectin synthesis by TGF-beta 1 appeared to imply a transcriptional event since it was no longer observed in the presence of DRB, a potent inhibitor of RNA polymerases, and because the level of fibronectin mRNA was stimulated under TGF-beta 1 treatment. Taken together, these results indicate that the increased expression of fibronectin is not closely related to growth-regulatory effects of TGF-beta 1 since it is also observed in adrenocortical cells, whose proliferation is unaffected by TGF-beta 1.
Adrenal Cortex↗
Characterization of the neuronal receptor for basic fibroblast growth factor and comparison to receptors on mesenchymal cells.
The receptor for basic fibroblast growth factor (bFGF) was characterized in highly enriched cultures of fetal hippocampal neurons. Two major components of binding could be distinguished. One component comprising about 70% of total binding was removed by 2 M NaCl, and by analogy to other cells could be presumptively attributed to glycosaminoglycans. The remaining 30% of binding which was resistant to 2 M NaCl reflects a high affinity receptor. Scatchard analysis indicated that the two components have Kd values of 500 and 120 pM, and densities of 165,000 and 35,000-60,000 sites/neuron, respectively. Cross-linking 125I-bFGF to neuronal cultures with disuccinimidyl suberate labeled a major membrane protein of 135 kDa and a minor protein of 85 kDa. Examination of neuronal cultures derived from multiple brain regions and membrane preparations from fetal brain suggested that the larger protein was widely distributed. The neuronal bFGF receptor was not blocked by heparin at concentrations up to 100 micrograms/ml. Twelve synthetic peptide fragments of bFGF were examined to determine the domain of bFGF interacting with the neuronal receptor. Inhibition was observed chiefly with a peptide including the sequence 103-146. The properties of the neuronal bFGF receptor were compared directly to those of the receptors characterized on BHK cells and other mesenchymal cells. Specific differences observed between neuronal and mesenchymal bFGF receptors are discussed.
Animals↗
Basic fibroblast growth factor is a substrate for protein phosphorylation and is phosphorylated by capillary endothelial cells in culture.
A phosphorylated basic fibroblast growth factor (FGF) can be detected in extracts of bovine capillary endothelial cells and human hepatoma cells. Accordingly, human basic FGF contains consensus sequences that account for its phosphorylation on Thr-112 by the catalytic subunit of the cAMP-dependent protein kinase A (PK-A) and on Ser-64 by the calcium- and phospholipid-dependent protein kinase C (PK-C). A kinetic analysis of both of these reactions revealed that basic FGF is among the better substrates for these enzymes. Although the kinase responsible for the phosphorylation in vivo has not yet been identified, we examined the effects of phosphorylation on the biological activity, heparin-binding capacity, and receptor-binding capacity of phosphorylated basic FGF. No effects of phosphorylation were observed when the mitogen was phosphorylated by PK-C. In contrast, when basic FGF was phosphorylated in the receptor-binding domain with PK-A, the growth factor was 3-8 times better at displacing radiolabeled basic FGF in the radioreceptor assay. No effects were seen on the binding of this FGF to immobilized heparin or cell-associated glycosaminoglycans, suggesting that this phosphorylation modifies the affinity of basic FGF for its receptor. Biological assays for basic FGF failed to identify differences between the phosphorylated and unphosphorylated forms of recombinant basic FGFs presumably because of the presence of ectophosphatases and the experimental conditions of proliferation and mitogenic assays (37 degrees C, 24-96 hr). Because the relative affinity of basic FGF for its receptor and cell-associated glycosaminoglycans may regulate its activity, the identification of a modified form of basic FGF may be of particular importance in understanding the mechanisms that regulate its biological activity, bioavailability, and processing to and from the extracellular matrix.
Animals↗
Differential effects of heparin, fibronectin, and laminin on the phosphorylation of basic fibroblast growth factor by protein kinase C and the catalytic subunit of protein kinase A.
Basic fibroblast growth factor (FGF) is synthesized as a phosphoprotein by both bovine capillary endothelial and human hepatoma cells in culture. Because basic FGF is characterized by its high affinity for heparin and its association in vivo with the extracellular matrix, we examined the possibility that the phosphorylation of this growth factor by purified protein kinase C (PK-C) and the catalytic subunit of cAMP-dependent protein kinase-A (PK-A) can be modulated by components of the extracellular matrix. Heparin and other glycosaminoglycans (GAGs) inhibit the ability of PK-C to phosphorylate basic FGF. In contrast, heparin can directly increase the phosphorylation of basic FGF by PK-A. While fibronectin, laminin, and collagen IV have no effect on the ability of PK-C to phosphorylate basic FGF, they all can inhibit the effects of PK-A. Thus, there is a differential effect of extracellular matrix-derived proteins and GAGs on the phosphorylation of basic FGF. The enhanced phosphorylation of basic FGF that is mediated by heparin is associated with a change in the kinetics of the reaction and the identity of the amino acid targeted by this enzyme. The amino acids that are targeted by PK-C and PK-A have been identified by phosphopeptide analyses as Ser64 and Thr112, respectively. In the presence of heparin, basic FGF is no longer phosphorylated by PK-A at the usual PK-A consensus site (Thr112), but instead is phosphorylated at the canonical PK-C site (Ser64). Accordingly, heparin inhibits the phosphorylation of basic FGF by PK-C presumably by masking the PK-C dependent consensus sequence surrounding Ser64. Thus, when basic FGF is no longer phosphorylated by PK-A in the receptor binding domain (Thr112), it loses the increased receptor binding ability that characterizes PK-A phosphorylated basic FGF. The results presented here demonstrate three novel features of basic FGF. First, they identify a functional effect of the binding of heparin to basic FGF. Second, they establish that the binding of heparin to basic FGF can induce structural changes that alter the substrate specificity of protein kinases. Third, and perhaps most important, the results demonstrate the existence of a novel interaction between basic FGF, fibronectin, and laminin. Although the physiological significance of this phosphorylation is not known, these results clearly suggest that the biological activities of basic FGF are regulated by a complex array of biochemical interactions with the proteins, proteoglycans, and glycosaminoglycans present in the extracellular milieu and the cytoplasm.
Amino Acid Sequence↗
Glycosylation of the basic fibroblast growth factor receptor. The contribution of carbohydrate to receptor function.
We have examined the glycosylation of the basic fibroblast growth factor (bFGF) receptor to determine whether carbohydrates contribute to receptor structure and function. Using a combination of cross-linking and radioreceptor assays, we demonstrated that the two bFGF receptors in baby hamster kidney cells have protein cores of 100 and 125 kDa. They are glycosylated to high mannose forms of 115 and 140 kDa and further processed to their mature forms of 130 and 150 kDa. Because peptide:N-glycosidase F, but not endo-alpha-N-acetylgalactosamidase can reduce the size of the bFGF receptors, the carbohydrate residues of the receptor appear all N-linked. The inability of deglycosylated receptors to bind 125I-bFGF supports the notion that the carbohydrate residues are required for receptor function. Furthermore, the capacity of the wheat germ agglutinin lectin to inhibit 125I-bFGF binding and the biological activity of bFGF suggests that N-acetylglucosamine residues are functionally significant components of the receptor.
Alkaloids↗
Bovine adrenocortical cells exhibit high affinity transforming growth factor-beta receptors which are regulated by adrenocorticotropin.
Transforming growth factor beta (TGF-beta) at picomolar concentrations has been previously shown to induce striking alterations of bovine adrenocortical cell differentiated functions, without detectable effect on growth activity (Feige, J.J., Cochet, C., and Chambaz, E. M. (1986) Biochem. Biophys. Res. Commun. 139, 693-700; Hotta, M., and Baird, A. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7795-7799). Adrenocortical cells in culture could bind 125I-labeled TGF-beta through at least two different binding systems. The highest affinity TGF-beta binding exhibited a Kd value of 5.7 X 10(-10) M and a calculated capacity of about 100,000 sites/cell, while the low affinity system yielded values of 4.3 X 10(-8) M and 2 X 10(6) sites/cell, respectively. The 125I-labeled TGF-beta bound to adrenocortical cells could be cross-linked using disuccinimidyl suberate and subsequent electrophoretic analysis revealed that TGF-beta was associated with two major cell components of about 280 kDa and 70-75 kDa, respectively, the latter one being resolved as a labeled doublet. Thus bovine adrenocortical cells exhibit a TGF-beta receptor similar to that defined by Massagué and co-workers (Cheifetz, S., Like, B., and Massagué, J. (1986) J. Biol. Chem. 261,9972-9978) in other cell types. Various growth factors, including fibroblast growth factor, as well as established hormonal activators of adrenocortical cell differentiated functions, such as angiotensin II and adrenocorticotropin, were examined as to their effect on TGF-beta receptor activity. A striking increase in the number of high affinity TGF-beta receptors was selectively elicited by ACTH in the nanomolar concentration range. This effect was time- and dose-dependent and was mimicked by cell treatment with dibutyryl cyclic AMP or forskolin. However, the ACTH-induced increase in receptor number was not impaired when protein synthesis was blocked. It is concluded that bovine adrenocortical cells are typical target cells for TGF-beta. This endocrine system represents a model in which, for the first time, the level of TGF-beta receptor is shown to be under hormonal regulation through a cyclic AMP-dependent pathway.
Adrenal Cortex↗
Temperature-sensitive Chinese hamster cell mutant with a defect in glycoprotein synthesis: accumulation of the EGF receptor in the endoplasmic reticulum and the role of the glucose-regulated protein GRP78.
A temperature-sensitive mutant of Chinese hamster fibroblasts with a defect in glycoprotein synthesis is investigated after transfection and amplification of the gene for the human EGF receptor. We demonstrate that at the nonpermissive temperature a partially glycosylated species of the receptor accumulates in the endoplasmic reticulum. The oligosaccharides present are the high mannose types, since they can be removed completely by treatment with endoglycosidase H. Pulse-chase experiments show that the abnormal species of the receptor cannot be chased to a form that is either resistant to endoglycosidase H, or altered in its mobility on SDS polyacrylamide gels. The abnormal species of the receptor appears within the first hour of a shift to the nonpermissive temperature, and no further changes are observed upon prolonged incubation of cells at 40 degrees C. However, after 3-4 hours immunoprecipitations of the receptor yield another protein, which has properties very similar, if not identical, to the glucose-regulated protein GRP78. The induction of this protein at 40 degrees C can be suppressed completely with an inhibitor of RNA synthesis, without any effect on the glycosylation defect, or on the accumulation of the EGF receptor in the endoplasmic reticulum.
Animals↗
Type beta transforming growth factor affects adrenocortical cell-differentiated functions.
Type beta transforming growth factor (TGF-beta) had no detectable effect on mitogenic activities of bovine adrenocortical cells in culture. However, the presence of TGF-beta (1 ng/ml) in the medium resulted in a striking alteration of adrenocortical cell steroidogenic activities, maximally expressed after 18-20 h of treatment. TGF-treated cells exhibited a basal as well as an adrenocorticotropin-stimulated cortisol production inhibition by an average 50-60%, while cAMP accumulation in response to the hormone was not modified. Detailed study of the adrenocortical steroid biosynthetic pathway by high performance liquid chromatography analysis and supply of representative steroid substrates revealed a drastic loss (average 50%) of the steroid 17 alpha-hydroxylase activity following TGF treatment. TGF-beta thus appeared as a potent negative modulator of adrenocortical 17 alpha-hydroxylase activity. This TGF-induced loss in the activity of a key steroidogenic enzyme resulted in a shift of the adrenocortical cell secretion pattern at the expense of the 17 alpha-hydroxysteroid end products. This 17 alpha-hydroxylation alteration was also expressed when TGF-treated cells were challenged by angiotensin II. However, in this case, an additional lesion was suggested by a 70-90% inhibition in angiotensin II-activated cortisol production. This could be explained by the observation that TGF-beta exposure induced an average 50% decrease in the adrenocortical cell angiotensin II receptor number without any detectable change in receptor affinity (Ka approximately 10(9) M-1). In addition, a parallel alteration in the angiotensin II-activated phosphoinositide breakdown was observed in TGF-treated cells, indicating that TGF-beta appears as a negative effector of the adrenocortical cell transmembrane signaling system in the case of angiotensin II. It is concluded that, in vitro, TGF-beta is a potent modulator of differentiated adrenocortical cell functions, in which at least two major negatively regulated specific targets were characterized. The mechanism(s) of action and the possible physiological significance of TGF-beta in the control of the development and the differentiated functions of the adrenocortical gland in vivo remain to be established.
Adrenal Cortex↗
Analysis of the protein glycosylation defect of a temperature-sensitive cell cycle mutant by the use of mutant cells overexpressing the human epidermal growth factor receptor after transfection of the gene.
A temperature-sensitive mutant with a defect in glycoprotein synthesis and a cell cycle (G1)-specific arrest at the nonpermissive temperature (Tenner et al., J. Cell. Physiol., 90:145-160, 1977; Tenner and Scheffler, J. Cell. Physiol., 98:251-266, 1979) was investigated further after a human epidermal growth factor (EGF) receptor gene had been transfected and amplified in these cells. While a temperature shift-up lead to an immediate arrest in the biosynthesis of mature EGF receptor and its appearance on the plasma membrane, the observed turnover of the preexisting receptor was too slow to account for the arrest of DNA synthesis in these mutant cells. Tunicamycin could in fact mimic the effect of a temperature shift on the biosynthesis of EGF receptor, but it did not have the same rapid effect on DNA synthesis and cell cycle progression. These mutants have also been shown to induce a set of stress proteins or glucose-regulated proteins, GRPs (Lee et al., J. Cell. Physiol., 129:277-282, 1986). The question is addressed whether the defect in glycoprotein synthesis is the primary defect and a possible cause of the induction of the GRPs, or whether a more basic defect at the level of the endoplasmic reticulum (ER) is responsible for the complex phenotype of the mutant. Our results argue in favor of a primary defect which indirectly affects N-linked glycosylation of proteins, as well as several other functions associated with the ER. We hypothesize that the defect affects the calcium distribution between ER and cytosol, since the calcium ionophore A23187 has an effect similar to that of a temperature shift.
Animals↗
Membrane receptors with protein-tyrosine kinase activity.
Protein-tyrosine kinase activities have appeared so far to be intrinsic for two classes of proteins: the transforming proteins of certain retroviral oncogenes and the membrane receptors for certain cellular growth factors. In this latter family, the protein-tyrosine kinase is activated upon binding of the growth factor to its receptor and phosphorylates both the receptor itself and other cell target proteins. Growth factor receptors are transmembrane glycoproteins able to undergo not only autophosphorylation but also phosphorylation by other protein kinases (e.g., protein kinase C). Both autophosphorylation and heterologous phosphorylation of the receptor are regulatory events for the ligand binding and protein-tyrosine kinase intrinsic activities of the growth factor receptors.
Animals↗
Type beta transforming growth factor is a potent modulator of differentiated adrenocortical cell functions.
Whereas TGF-beta exhibited no detectable effect on DNA synthesis, it was found to exert a striking inhibitory effect on the steroidogenic activities of bovine adrenocortical cells in culture. Basal, as well as ACTH- and angiotensin II- activated adrenocortical cortisol productions were inhibited in a time and dose-dependent manner following TGF-beta treatment. Half-maximum inhibition of ACTH- and AII-activated steroidogenesis was observed with TGF-beta concentrations of 0.40 and 0.12 ng/ml, respectively. This effect was half maximal after 6 hours of cell exposure to optimally effective TGF-beta concentrations (1 ng/ml) and reached a plateau after 12-15 hours, resulting in an average 60% inhibition in the steroidogenic response to ACTH and 90% in the case of AII. Supply of different exogenous steroid substrates to support steroidogenesis in adrenocortical cells pointed to a marked loss in steroid-17 alpha hydroxylase activity as a major alteration following TGF-beta treatment. TGF-beta thus appears as a potent modulator of differentiated adrenocortical cell functions in vitro; in this regard it may play a significant role in the development and the regulation of adrenal cortex in vivo.
17-alpha-Hydroxypregnenolone↗