The type I insulin-like growth factor receptor is a motility receptor in human melanoma cells

Insulin-like growth factors I and II (IGF-I and II) and insulin are chemotactic agents for the human melanoma cell line A2058. As shown in this report, the motility receptor mediating this response is the heterodimeric type I IGF receptor. These three factors are able to compete with 125I-labeled IGF-I for binding to the cell surface with IC50 values equal to approximately 2 (IGF-I), approximately 150 (IGF-II), and approximately 300 nM (insulin). Cross-linking of 125I-IGF-I to the cell surface with disuccinimidyl suberate followed by analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography reveals a 130-kDa protein (reduced) consistent with the alpha component of a type I receptor and a 38-kDa protein which does not bind insulin, and thus could be another IGF-I cell surface binding protein. The anti-IGF-I receptor monoclonal antibody (alpha IR-3) also competes with labeled IGF-I in binding experiments. In contrast, a control monoclonal antibody, matched to alpha IR-3 with respect to IgG subclass, has no significant effect on IGF-I binding. While alpha IR-3 inhibits the motility induced by IGF-I, IGF-II, and insulin, pertussis toxin (0.01-1.0 micrograms/ml) has no significant effect on the motility induced by the insulin-like growth factors or insulin on this cell line. Therefore, the type I IGF receptor appears to mediate a highly potent pertussis toxin-insensitive motility response to IGF-I, IGF-II, and insulin. In contrast, motility induced by the autocrine motility factor, a cytokine produced by the A2058 cells, is not affected by alpha IR-3 but is extremely sensitive to pertussis toxin. When mixtures of autocrine motility factor and IGF-I are employed to induce chemotaxis, the resulting motility is greater than that induced by either agent alone. These data indicate that motility in this melanoma cell line can be initiated through multiple receptors that stimulate the cells by separate transduction pathways. This capability to respond to multiple stimuli could enhance the metastatic potential.     

Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout

The relative function of IGF-I and insulin on fish muscle metabolism and growth has been investigated by the isolation and culture at different stages (myoblasts at day 1, myocytes at day 4, and myotubes at day 10) of rainbow trout muscle cells. This in vitro model avoids interactions with endogenous peptides, which could interfere with the muscle response. In these cells, the effects of IGF-I and insulin on cell proliferation, 2-deoxyglucose (2-DG), and l-alanine uptake at different development stages, and the use of inhibitors were studied and quantified. Insulin (10-1,000 nM) and IGF-I (10-100 nM) stimulated 2-DG uptake in trout myocytes at day 4 in a similar manner (maximum of 124% for insulin and of 142% for IGF-I), and this stimulation increased when cells differentiated to myotubes (maximum for IGF-I of 193%). When incubating the cells with PD-98059 and especially cytochalasin B, a reduction in 2-DG uptake was observed, suggesting that glucose transport takes place through specific facilitative transporters. IGF-I (1-100 nM) stimulated the l-alanine uptake in myocytes at day 4 (maximum of 239%), reaching higher values of stimulation than insulin (100-1,000 nM) (maximum of 160%). This stimulation decreased when cells developed to myotubes at day 10 (118% for IGF-I and 114% for insulin). IGF-I (0.125-25 nM) had a significant effect on myoblast proliferation, measured by thymidine incorporation (maximum of 170%), and required the presence of 2-5% fetal serum (FBS) to promote thymidine uptake. On the other hand, insulin was totally ineffective in stimulating thymidine uptake. We conclude that IGF-I is more effective than insulin in stimulating glucose and alanine uptake in rainbow trout myosatellite cells and that the degree of stimulation changes when cells differentiate to myotubes. IGF-I stimulates cell proliferation in this model of muscle in vitro and insulin does not. These results indicate the important role of IGF-I on growth and metabolism of fish muscle.     

Insulin-like growth factor I (IGF-I) production and the presence of IGF-I receptors in rat medullary thyroid carcinoma cell line 6-23 (clone 6)

To clarify whether insulin-like growth factor I (IGF-I) is an autocrine growth factor of rat medullary thyroid carcinoma (MTC) cell line, 6-23 (clone 6), IGF-I binding to MTC cell membranes, IGF-I levels in the conditioned culture medium of MTC cells and the effects of IGF-I on methyl-[3H]thymidine incorporation to MTC cells were examined. Scatchard analysis of saturation binding studies revealed the association constant and the maximal binding capacity were 1.0 x 10(9) M-1 and 199 fmol/mg of membrane protein, respectively. The binding of [125I]IGF-I to MTC cell membranes was inhibited by unlabeled IGF-I, IGF-II and insulin; the relative potencies were IGF-I greater than IGF-II much greater than insulin, suggesting the presence of type I IGF receptors in MTC cells. IGF-I levels in the conditioned culture medium of MTC cells were 120 +/- 3 pM (mean + SE). IGF-I (10(-10) to 10(-8) M) dose-dependently stimulated methyl-[3H]thymidine incorporation to MTC cells. These findings suggest a possible role of IGF-I as an autocrine growth factor for MTC cells.     

Multihormonal regulation of insulin-like growth factor-I-related protein in MCF-7 human breast cancer cells

MCF-7 human breast cancer cells have been studied for hormonal regulation of secretion of an insulin growth factor-I (IGF-I)-related growth factor. 17 beta-Estradiol, which is required for tumorigenesis of the cell line in the nude mouse and which stimulates proliferation in vitro, was able to significantly induce IGF-I secretion at 10(-13) M, with maximal induction at 10(-11) M. Under optimal conditions IGF-I could be induced 4-fold after 4 days. Demonstration of estrogenic stimulations required removal of phenol red, a weak estrogen, from the cell culture medium. In addition to estrogen, insulin, epidermal growth factor, and transforming growth factor alpha induce both cellular proliferation and IGF-I secretion, while growth inhibitory antiestrogens, transforming growth factor beta, and glucocorticoids have the opposite effect. In each case, modulations in IGF-I secretion preceeded effects on cellular proliferation. IGF-I was not regulated by human GH, basic fibroblast growth factor, platelet-derived growth factor, or PRL, none of which affected proliferation rate. Thus, regulation of IGF-I secretion in human breast cancer is controlled by different hormones from those previously reported in human fibroblasts. Regulation of IGF-I by neither estrogen nor antiestrogen was associated with changes in steady-state mRNA levels; thus regulation may occur at a step beyond mRNA. We conclude that IGF-I production is tightly coupled to growth regulation by estrogens, antiestrogens, and other hormones and may contribute to autocrine and/or paracrine growth regulation by these agents in breast cancer.     

Synergistic action of growth hormone and insulin-like growth factor I (IGF-I) on proliferation and estradiol secretion in porcine granulosa and theca cells cultured alone or in coculture

The effect of growth hormone (GH) on insulin-like growth factor I (IGF-I) secretion and the effects of GH and IGF-I on [(3)H] thymidine incorporation and estradiol (E2) secretion by theca interna (Tc) and granulosa cells (Gc) cultured alone and in coculture were studied in cultured porcine follicular cells, prepared from small (SF), medium (MF) and large (LF) preovulatory follicles. We demonstrated that both Tc and Gc secrete IGF-I and that GH had no effect on IGF-I secretion by Tc but, increased IGF-I secretion by Gc isolated from SF and cultured alone or in coculture. IGF-I stimulated secretion of E2 by all cells, except in Tc derived from SF in which the effect was not statistically significant. The only stimulatory effect of concurrent treatment with GH on E2 secretion was noted in Tc derived from MF. IGF-I increased the [(3)H] thymidine incorporation in all Tc cells but GH did not augment this effect. In Gc, IGF-I stimulated [(3)H] thymidine incorporation in cells derived from SF and MF but not from LF. GH had no stimulatory effect except on Gc derived from LF and grown alone. The highest stimulatory effect was observed in SF. This was smaller in MF and no effect was noted in LF. In conclusion, our work shows that both Gc and Tc are sites of IGF-I production and for the first time shows the stimulatory role of IGF-I in proliferation of Tc cells derived from all types of follicles and augmentation of E2 secretion in Tc derived from MF and LF. The promotion of the mitogenic activity in Tc by IGF-I during all stages of follicular development suggests an important role for theca cells in follicular growth.     

Comparative studies on the effects of steroidal and nonsteroidal oestrogen antagonists on the proliferation of human breast cancer cells

Pure and partial agonist antioestrogens, exemplified by ICI 164,384 and tamoxifen or 4-hydroxytamoxifen (4'-OHT) respectively, differ in their capacity to inhibit the growth of MCF-7 human breast cancer cells. Under basal conditions which maintain but do not permit proliferation of MCF-7 cells, growth rate was enhanced by oestradiol, Phenol Red, insulin and 4'-OHT but not by ICI 164,384. Oestradiol and insulin together enhanced cell growth rate synergistically. 4'-OHT, but not ICI 164,384, similarly increased insulin-stimulated cell growth in the absence of oestradiol. ICI 164,384 blocked the stimulatory action of 4'-OHT. The stimulatory effect of the peptide growth factors TGF-alpha and IGFI on MCF-7 cells were attenuated by ICI 164,384 and 4'-OHT; ICI 164,384 was more effective than 4'-OHT. The antiproliferative action of ICI 164,384 on oestradiol or growth factor stimulated MCF-7 cells was only weakly inhibited by TGF-beta antibodies.     

Different action of ovine GH on porcine theca and granulosa cells proliferation and insulin-like growth factors I- and II-stimulated estradiol production

Growth hormone (GH) and insulin-like growth factors (IGFs) are recognized as regulators of ovarian function. This study was designed to compare the effect of GH and IGFs added alone or together on porcine theca interna and granulosa cells proliferation and steroidogenesis. Moreover, the effect of GH on IGF-I secretion was examined. Cells were isolated from medium size follicles and cultured in vitro for 48 h in serum free medium. Estradiol and IGF-I medium concentrations were determined by radioimmunoassays. Proliferation was evaluated by alamar blue assay and by radiolabelled thymidine incorporation. GH increased IGF secretion by granulosa cells while decreased its secretion by theca cells. Proliferation of both cell types was stimulated by IGF-I and IGF-II (30 ng/ml) and modestly inhibited by GH (100 ng/ml). Insulin-like growth factor II increased, in a statistically significant manner, estradiol secretion by both cell types, while IGF-I stimulated estradiol secretion to a greater extent by granulosa then by theca cells. The synergistic action of GH and IGFs on estradiol secretion was stimulatory in theca cells and inhibitory in granulosa cells. These data demonstrate that despite its direct action on estradiol secretion by granulosa and theca cells, GH also modulated estradiol secretion induced by IGFs. Differences in the estradiol production in response to GH alone and the effect of the synergistic action of GH and IGFs suggest that different cellular mechanisms for these hormones are triggered in each cell type.     

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.     

Human granulosa cells in vitro: characteristics of growth, morphology and influence of some cytokines on steroidogenesis.

Granulosa cells (GCs) were characterised morphologically by light and electron microscopy. The steroidogenic capability of GCs in vitro was estimated by radioimmunoassay (RIA): oestradiol (E2), progesterone (P) and androstenedione (A) secreted into the culture medium were measured. The influence of several culture media and anchorage of the cell either to plastic vessels (monolayer) or to collagen fibrils (in gel) were studied. As the various culture media were assayed with regard to their suitability for IVF, it was found that Ham's F10 is quite satisfactory (in agreement with other observations on embryo cultures). A chemically defined medium BM 86 was found to be inadequate. In addition to the two cell types which are known, a third cell type which can perform efficient aromatisation (E2 production) in vitro is characterised here. The influence of cytokines/growth factors (GF) like insulin-like GF (IGF-1), epidermal GF (EGF), platelet-derived GF (PDGF) and fibroblast GF (FGF) on steroidogenesis was tested either alone or with human chorionic gonadotrophin (hCG). Except for oestradiol (E2) from early GCs, hCG generally stimulated progesterone (P) and E2 secretion. EGF by itself enhanced the secretion of P but not of E2. EGF did not affect hCG stimulation of P, but reduced that of E2. In contrast, in pre-ovulatory GCs IGF-I reduced the stimulatory effect of hCG on both E2 and P. In early GCs IGF-I potentiated hCG stimulation of P. In early GCs, neither hCG nor IGF-I nor a combination of IGF-I with hCG had any effect on E2 production.     

Cycloheximide blocks insulin-like growth factor I but not somatostatin inhibition of growth hormone secretion

Growth hormone secretion is controlled by the two hypothalamic hormones, growth hormone releasing factor (GRF) and somatostatin. In addition, the insulin-like growth factors (IGF or somatomedins) which are themselves growth hormone dependent, inhibit growth hormone release in vitro, therefore acting to close the negative feedback loop. The studies reported here examine some of the differences between inhibition of growth hormone secretion by somatostatin and IGF-I in vitro. The major finding is that cycloheximide, a protein synthesis inhibitor, blocks inhibition of GRF-stimulated growth hormone release caused by IGF-I, without changing the inhibition caused by somatostatin. The experiments were done by exposing mixed rat adenohypophysial cells to secretagogues with or without cycloheximide for 24 h in a short term culture. Somatostatin (0.6 nM) totally blocked rat GRF (1 nM) stimulated growth hormone release to values 48% of control (nonstimulated values), while IGF-I (27 nM) only reduced the GRF-stimulated growth hormone release by 27 +/- 3% (N = 5). Cycloheximide (15 micrograms/mL) totally blocked the effect of IGF-I but not somatostatin. A low concentration (0.12 nM) of somatostatin, which only partly inhibited growth hormone release, was also unaffected by cycloheximide. In purified rat somatotrophs, somatostatin (0.1 nM) inhibited GRF-stimulated cAMP levels slightly and reduced growth hormone release while IGF-I (40 nM) had no effect. We suggest that IGF-I inhibits only the secretion of newly synthesized growth hormone, while somatostatin inhibits both stored and newly synthesized growth hormone pools.     

Direct evidence that the insulin receptor mediates a mitogenic response in cultured human fibroblasts

To define the role of the insulin receptor in mediating a mitogenic response in cultured human fibroblasts, the effects of specific monoclonal antibodies against the insulin and the type I IGF receptor on insulin-stimulated [3H]thymidine incorporation were investigated. Insulin stimulated [3H]thymidine incorporation in a biphasic fashion. In the first phase, a half-maximal effect was observed at 20 ng/ml, and a seemingly maximal effect was obtained at 100-1000 ng/ml. With 10 micrograms/ml insulin, a secondary increase in [3H]thymidine incorporation was seen which was similar to the maximal effect of IGF-I. These [3H]thymidine incorporation results were corroborated with cell replication studies. MC-51, a highly specific monoclonal antibody for the insulin receptor, inhibited the stimulation of [3H]thymidine incorporation by 25 ng/ml of insulin. AlphaIR-3, a monoclonal antibody specifically directed against the type I IGF receptor, had no significant effect on insulin-stimulated [3H]thymidine incorporation at low (10-1000 ng/ml) concentrations of insulin. However, alpha IR-3 interfered with the incremental increase in [3H]thymidine incorporation observed at 10-100 micrograms/ml insulin. These data demonstrate that insulin, at low concentrations, is capable of stimulating DNA synthesis and replication of human fibroblasts through interaction with its own receptor, while at supraphysiological concentrations, much of insulin's mitogenic effect is mediated through the type I IGF receptor.     

Activin stimulates proliferation of rat ovarian thecal-interstitial cells

There is growing evidence that the function of ovarian theca-interstitial (T-I) cells may be modulated by paracrine actions of activin, inhibin, and follistatin. Furthermore, either dysregulation, dysfunction, or both, of these peptides may play a role in conditions associated with T-I hyperplasia, such as polycystic ovary syndrome (PCOS) and hyperthecosis. This study was designed to evaluate the role of activin, inhibin, and follistatin in the modulation of T-I cell proliferation. Interaction of these peptides with insulin-like growth factor-I (IGF-I), a known stimulator of T-I cell proliferation, was also assessed. Purified rat T-I cells were cultured for 48 h in chemically defined media and with or without activin (3-30 ng/ml), inhibin (3-30 ng/ml), follistatin (100 ng/ml), and/or IGF-I (10 nM). T-I cell proliferation was assessed using radiolabeled thymidine incorporation assay. Activin alone stimulated proliferation of T-I cells in a dose-dependent fashion (by up to 320% above control; P < 0.001), whereas inhibin alone or follistatin alone had no significant effect. Inhibin had also no effect on activin-induced proliferation. Follistatin significantly reduced the stimulatory effects of activin and decreased proliferation by up to 46% (P < 0.01) below the level attained in the presence of activin alone. IGF-I (10 nM), at a dose producing a near-maximal effect, increased proliferation by 175% above control (P < 0.001); insulin (10 nM) increased proliferation by 52% above control (P < 0.03). A combination of IGF-I (10 nM) and activin (30 ng/ml) resulted in a 1090% increase of proliferation above control (P < 0.001); this stimulatory effect was significantly greater than that achieved in the presence of either activin alone or IGF-I alone (P < 0.001). Similarly, a combination of insulin (10 nM) and activin (30 ng/ml) increased proliferation by 506% above control levels. Flow cytometry evaluation revealed that activin increased the proportion of actively dividing cells (in S or G2/M phase of the cell cycle) by 42% (P < 0.02), whereas IGF-I had no effect on the proportion of actively dividing cells. The present findings indicate that an activin-follistatin system may be involved in the regulation of the size of ovarian thecal-stromal compartment. In view of the synergy between activin and IGF-I, and the difference in the effects on the cell cycle distribution, stimulation of T-I proliferation by these agents is likely to be mediated via separate transduction pathways. Excess activin or insufficient follistatin may contribute to T-I hyperplasia.     

Hormonal regulation of insulin-like growth factor I secretion by bovine adrenal cells

The role of insulin-like growth factor I (IGF-I) on the specific function of several steroidogenic cells has been recently reported. Since IGF-I is produced by several tissues, we have investigated whether bovine adrenal cells secrete this peptide. Purification of conditioned medium from adrenal cells incubated with [35S]methionine through affinity chromatography (monoclonal anti-IGF-I antibody), high pressure liquid chromatography, and polyacrylamide gel electrophoresis revealed a single band of similar Mr as pure recombinant IGF-I. Moreover, the purified adrenal-secreted IGF-I displaced bound 125I-IGF-I to its adrenal receptors, and pretreatment of adrenal cells with the purified peptide enhanced the acute corticotropin (ACTH)-induced cAMP production as recombinant IGF-I. The basal secretion of IGF-I (6 +/- 1 ng/48 h/10(6) cells) was stimulated 3-, 4.5-, and 9.5-fold by fibroblast growth factor, angiotensin II (A-II), and ACTH, respectively, but not by growth hormone. The stimulatory effects of A-II and ACTH were dose-dependent (ED50 congruent to 2.5 x 10(-8) and 1.5 x 10(-10) M, respectively), and the effects of both hormones were additive. Glucocorticoids were not the mediators of the effect of the two hormones on IGF-I secretion, since inhibition of their steroidogenic action by aminoglutethimide did not significantly modify IGF-I secretion. An immunoreactive IGF-I material was also secreted by mouse adrenal tumor cell line Y-1, but the stimulatory effect of ACTH was only 2-fold, and there was no effect of A-II. Since bovine adrenal cells contain specific IGF-I receptors and this peptide is required for the maintenance of some adrenal cell-specific function, the present data suggest that IGF-I may act in an autocrine fashion to stimulate adrenal cell differentiation stimulated by ACTH and A-II.     

Effect of insulin receptor down regulation on insulin-stimulated thymidine incorporation in cultured human fibroblasts and tumor cell lines.

Insulin receptors in transformed tissue are relatively resistant to down regulation by insulin, and although receptor downregulation reduces rapid onset biologic responses to insulin in normal tissue, this is not observed in tumor cells. The present study compares longterm insulin responses (thymidine incorporation and cell growth) in normal human fibroblasts with responses in human tumor cell lines (MCF-7, T-47D and HCT-8) to determine whether these responses are also resistant to the effects of receptor down regulation. Thymidine incorporation into fibroblasts was more responsive to insulin than was incorporation into tumor cells, although stimulation of uptake into fibroblasts was not paralleled by changes in cell replication. In contrast, physiological insulin concentrations inhibited, and high concentrations of insulin stimulated, thymidine incorporation and cell replication in MCF-7 and T-47D cells. All insulin concentrations inhibited thymidine incorporation in HCT-8 cells without affecting cell replication. The responsiveness of fibroblasts, MCF-7 and HCT-8 cells to insulin was unaltered by down regulation of insulin receptors prior to measuring thymidine incorporation, whereas receptor down regulation paradoxically increased the responsiveness of T-47D cells to insulin. Exposure of fibroblasts to 5 x 10(-8) M dexamethasone for 24h increased their responsiveness to insulin but did not influence the response of MCF-7 or HCT-8 cells, whereas insulin-stimulated incorporation of thymidine in T-47D cells was inhibited. Thus, receptor down regulation does not influence the longterm biologic response to insulin in normal cells, and paradoxically increases responsiveness in one of three tumor cell lines. These changes may contribute to the well-described stimulatory effects of insulin on tumor cell growth and inhibition of this response with dexamethasone may be relevant to cancer treatment programs.     

The signaling potential of the receptors for insulin and insulin-like growth factor I (IGF-I) in 3T3-L1 adipocytes: comparison of glucose transport activity, induction of oncogene c-fos, glucose transporter mRNA, and DNA-synthesis.

The receptors for insulin and insulin-like growth factor I (IGF-I) have in common a high sequence homology and diverse overlapping functions, (e.g., the stimulation of acute metabolic events and the induction of cell growth.). In the present study, we have compared the potential of insulin and IGF-I receptors in stimulating glucose transport activity, glucose transporter gene expression, DNA-synthesis, and expression of proto-oncogene c-fos in 3T3-L1 adipocytes which express high levels of both receptors. Binding of both hormones to their own receptors was highly specific as compared with binding to the respective other receptor (insulin receptor: KD = 3.6 nM, KI of IGF-I greater than 500 nM; IGF-I receptor, KD = 1.1 nM, KI of insulin = 191 nM). Induction of proto-oncogene c-fos mRNA by insulin and IGF-I paralleled their respective receptor occupancy and was thus induced by both hormones via their own receptor (EC50 of insulin, 3.7; IGF-I, 3.9 nM). Similarly, both insulin and IGF-I increased DNA synthesis (EC50 of insulin, 5.8 nM; IGF-I, 4.0 nM), glucose transport activity (EC50 of insulin, 1.7 nM; IGF-I, 1.4 nM), and glucose transporter (GLUT4) mRNA levels in concentrations corresponding with their respective receptor occupancy. These data indicate that in 3T3-L1 cells the alpha-subunits of insulin and IGF-I receptors have an equal potential to stimulate a metabolic and a mitogenic response.     

Glucose-induced translational control of proinsulin biosynthesis is proportional to preproinsulin mRNA levels in islet beta-cells but not regulated via a positive feedback of secreted insulin

Proinsulin biosynthesis is regulated in response to nutrients, most notably glucose. In the short term (/=10-fold). Importantly, neither exogenously added nor secreted insulin were found to play any role in regulating insulin secretion, proinsulin translation, preproinsulin mRNA levels, or total protein synthesis. The results presented here indicate that long term nutritional state sets the preproinsulin mRNA level in the beta-cell at which translation control regulates short term changes in rates of proinsulin biosynthesis in response to glucose, but this is not mediated by any autocrine effect of insulin.     

Regulation of growth of LNCaP human prostate tumor cells by growth factors and steroid hormones

The mitogenic activity of several growth factors on androgen responsive LNCaP human prostate tumor cells was studied. A two-fold stimulation of cell proliferation was observed after a culture period of 6 days in 1 ng EGF/ml, 10 ng TGF-alpha/ml or 20 ng basic FGF/ml. TGF-beta (0.02 ng/ml), which did not affect cell proliferation when added alone to the culture medium, inhibited the EGF- and TGF-alpha-induced growth. The synthetic androgen R1881 (0.1 nM) stimulated cell proliferation three-fold and increased the number of EGF receptors from 11500 to 28500 sites/cell. One of the mechanisms involved in androgen action on these cells is therefore an increased EGF receptor expression and increased sensitivity to EGF. TGF-beta did not directly affect androgen-responsive growth but inhibited the synergistic effect of EGF. A considerable expression of TGF alpha (precursors) could be demonstrated on the cells by immunohistochemical staining. However the staining intensity was not affected by androgens. These results make it less likely that androgen-responsive growth is mediated by regulation of secretion of an EGF- or TGF alpha-like activity, which in turn acts in an autocrine manner to stimulate growth. Estrogens, progestagens and antiandrogens do not inhibit androgen responsive growth of LNCaP cells but have striking growth stimulatory effects, increase EGF receptor level and increase acid phosphatase secretion. LNCaP cells contain a modified androgen receptor system with respect to both steroid specificity and antiandrogen sensitivity. It has recently been shown that the stimulatory effects are due to a mutated amino acid in the steroid binding domain of the androgen receptor.     

Response of porcine theca and granulosa cells to GH during short-term in vitro culture

In Experiment 1, the influence of exogenous GH on steroid secretion by granulosa and theca interna cells recovered from small (1-3 mm), medium (4-6 mm) and large (8-12 mm) follicles was tested. In the second experiment, theca cells (Tc) and granulosa cells (Gc) obtained from large follicles were cultured separately or in two types, Tc/Gc co-culture, where both types of cells were mixed in one well or Gc and Tc were separated by cell culture membrane inserts. In the third experiment, the influence of GH on the morphology of Gc and Tc cells and activity of Delta(5),3beta-hydroxysteroid dehydrogenase (3beta-HSD) was studied. Cells were grown in the control medium (M199+5% of calf serum) or supplemented with 100 ng/ml GH. Testosterone (10(-7) M) was added as the aromatase substrate to granulosa cells cultures. The media were assayed after 48 h of culture for progesterone and oestradiol by RIA. GH added to the culture media had no effect on oestradiol and progesterone secretion by granulosa cells isolated from small and medium follicles while it stimulated both oestradiol and progesterone secretion by Gc isolated from large preovulatory follicles. A stimulatory effect on oestradiol secretion by Tc isolated from all size follicles was observed. GH did not stimulate progesterone secretion by Tc isolated from small follicles but stimulated progesterone secretion by Tc isolated from medium and large preovulatory follicles. Both co-culture systems exhibited synergistic effect on oestradiol secretion. The stimulatory effect on progesterone secretion under the influence of GH was observed in Gc cultured alone and Tc cultured alone. In contrast, the secretion of progesterone was attenuated in both co-culture systems and the addition of GH further augmented this attenuation. A statistically significant increase in oestradiol secretion was observed in all culture conditions. The addition of GH to the culture medium stimulated the activity of 3beta-HSD compared with the control culture from both types of cells. In conclusion, the present studies indicate that there are direct and follicular development stage dependent actions of GH on steroidogenesis of porcine follicular cells.