Biomedical subjects

S Shimasaki

Publications and source records attributed to S Shimasaki.

At least 37 records · Page 2Linked to original sources

PubMed1997

Cellular distribution of insulin-like growth factor binding protein mRNAs and peptides during rat lung development.

A role for IGF binding proteins (IGFBPs) in lung development is suggested by the identification of IGFBPs in lung tissue and production of IGFBPs by fetal lung cells in culture. To characterize the expression of IGFBPs during lung development in the rat in vivo (16 days gestation through adulthood), the expression of IGFBP mRNAs (IGFBP-1 to IGFBP-6) was examined by Northern analysis and in situ hybridization, and IGFBP peptides (IGFBP-2, IGFBP-3, and IGFBP-5) were localized by immunohistochemistry. IGFBP-1 mRNA was not detectable. IGFBP-2 mRNA (1.8 kb) was expressed in both fetal and postnatal life with peak expression during the fetal pseudoglandular stage. IGFBP-2 mRNA was localized mainly to airway epithelium. IGFBP-3 mRNA (2.4 kb) was maximally expressed postnatally in the saccular stage of lung development; it was identified in airway epithelium and interstitium in the fetal lung, but predominantly in airway epithelium after birth. IGFBP-4 (2.6 kb) and IGFBP-5 (6.0 kb) mRNA levels were maximal after birth, from 3 to 21 days postnatal (saccular and alveolar stage). IGFBP-4 mRNA was localized primarily to the interstitium and blood vessels early in development, but was abundant in airway epithelium in the adult. IGFBP-5 mRNA was most abundant in the airway epithelium. IGFBP-3, IGFBP-4, IGFBP-5, and to a lesser extent IGFBP-6 were localized to the large cartilaginous airways in the adult. IGFBP-2, IGFBP-3, and IGFBP-5 peptides were distributed more widely than their respective mRNAs, with a temporal pattern of immunoreactivity following that of their mRNAs. Maximal staining was noted in airway epithelium for IGFBP-2 in the newborn, for IGFBP-3 in the saccular stage (newborn to 3 days postnatal), and for IGFBP-5 in the alveolar stage (5 to 21 days postnatal). Our studies demonstrate that IGFBP-2, IGFBP-3, IGFBP-4, and IGFBP-5 are synthesized and distributed in spatially and temporally different patterns in the developing lung. The widespread distribution of IGFBP immunoreactivity compared with their respective mRNAs suggests that IGFBPs are important paracrine factors in the regulation of IGF action in the developing lung.

Animals↗

PubMed1996

IGF-binding protein mRNAs in the human fetus: tissue and cellular distribution of developmental expression.

Insulin-like growth factors (IGF-I and IGF-II) are synthesized by most embryonic and fetal tissues, and regulate cellular growth and differentiation as autocrine/paracrine factors. A family of six IGF-binding proteins (IGFBPs) modulate IGF biological actions as both negative (inhibitory) and positive (potentiating) modulators. To determine the tissue distribution of IGFBP mRNA expression, we performed Northern blot analysis and in situ hybridization of human fetal tissues during gestational ages 10-16 weeks (n = 8). IGFBP-1 mRNA was expressed only in the liver, whereas other IGFBP mRNAs were expressed in variable abundance in every tissue examined. IGFBP-2 mRNA was expressed in moderate abundance in every tissue with the highest level observed in the liver; IGFBP-3 mRNA was expressed most abundantly in the skin, muscle and heart; IGFBP-4 mRNA was expressed in moderate abundance equally in all tissues; IGFBP-5 mRNA was expressed most abundantly in the skin, muscle and stomach, and IGFBP-6 mRNA was expressed in low abundance in all tissues. Notable exceptions were that liver expressed little or no IGFBP-4, -5 and -6 mRNAs, spleen and thymus expressed low levels of IGFBP-5 mRNA, and brain expressed little or no IGFBP-5 and IGFBP-6 mRNA. In situ hybridization of human fetal tissues showed IGFBP mRNAs were expressed in both epithelial and mesenchymal cells depending on the specific IGFBP and the stage of development. IGFBP-3, -4, and -5 mRNAs were localized mainly in the mesenchymal cells, and IGFBP-2 mRNA was localized predominantly in the epithelial cells. IGFBP-6 mRNA was localized in low abundance in both epithelial and mesenchymal cells. These studies indicate that IGFBPs are important paracrine modulators of IGF action on cellular growth and differentiation, by modulating IGF-dependent or IGF-independent actions.

Fetus↗

PubMed1995

Gonadotropin-releasing hormone overcomes follicle-stimulating hormone's inhibition of insulin-like growth factor-5 synthesis and promotion of its degradation in rat granulosa cells.

The effect of a gonadotropin-releasing hormone-agonist (GnRH-a) on the synthesis of insulin-like growth factor-binding protein-5 (IGFBP-5), a physiological marker for atresia, was investigated. Granulosa cells obtained from diethylstilbestrol (DES)-treated immature female rats were cultured in serum-free medium for 72 h with GnRH-a and the conditioned media were subjected to immunoblot analysis using rat IGFBP-5 specific antibody. GnRH-a caused a dose-dependent (ED50 = 8.6 x 10(-11) M) accumulation of IGFBP-5, which migrated as 35 (non-glycosylated) and 36 kDa (glycosylated) bands under reducing conditions. A maximally effective dose of GnRH-a (10(-9) M) caused a 4-fold increase in IGFBP-5 accumulation. In contrast, increasing doses of porcine follicle-stimulating hormone (pFSH) caused a biphasic effect on IGFBP-5 accumulation. A low dose of pFSH (0.25 ng/ml) increased and higher doses of pFSH (22.5 ng/ml) decreased the 35 and 36 kDa IGFBP-5 bands. In the presence of high doses of pFSH (20.75 ng/ml), a 22 kDa band corresponding to a cleaved IGFBP-5 fragment appeared in the media. When the granulosa cells were cultured with a saturating dose of pFSH, co-addition of GnRH-a dose dependently inhibited the FSH effects (ED50 = (2.3-3.7) x 10(-10) M). The GnRH-a effects were completely blocked by co-incubation with GnRH-antagonist. IGFBP-5 mRNA accumulation levels were increased by GnRH-a in a dose dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

The heparin binding site of follistatin is involved in its interaction with activin.

Whether the heparin-binding site of follistatin would interact with activin has been examined. When a mixture of recombinant human follistatin-288 (rhFS-288) and -315 (rhFS-315) was applied to an activin-coupled affinity column, followed by stepwise elution of the column using 4M urea, 8M urea, 1M guanidine-HCl and 2M guanidine-HCl, rhFS-315 was eluted with 4M urea, while rhFS-288 was eluted with 2M guanidine-HCl. This finding implies that the carboxylterminal 27 amino acid extension of rhFS-315, which is not present in rhFS-288, affects the binding of follistatin with activin. Addition of heparin (50 micrograms/ml) to the elution solvent caused rhFS-288 to elute with 4M urea, whereas rhFS-315 was not affected. These data suggest for the first time that these two structurally related follistatin molecules interact with activin by different modes of binding and, in the presence of heparin, the interaction of rhFS-288 with activin is indistinguishable from that of rhFS-315. Two analogs of rhFS-288 mutated at the heparin binding site were eluted with 8M urea or 1M guanidine-HCl, distinct from the elution profile of the intact rhFS-288. These results indicated that mutation at the heparin binding site alters the activin binding affinity. In addition, bioassay of the two mutants showed that they were less potent than the rhFS-288. These findings suggest that the heparin binding site of follistatin also contributes to its binding for activin, and heparin may play an important role in the bioactivity of follistatin.

Activins↗

PubMed1995

Follistatin concentrations in follicular fluid of normal and polycystic ovaries.

Follistatin (FS) is an activin/inhibin binding protein which is believed to act in an autocrine/paracrine manner to regulate growth and differentiation. Although FS has been identified in human follicular fluid, it remains unclear how its concentration changes during selection and atresia, and what the concentrations of FS are in follicles of women with polycystic ovary syndrome (PCOS). Towards this goal, we have measured by radioimmunoassay the concentrations of FS in follicular fluid obtained from dominant and atretic cohort follicles of normal cycling women, preovulatory follicles of in-vitro fertilization (IVF) patients, and small Graafian follicles of patients with PCOS. In all cases, the follicular fluid concentration of FS was much higher (approximately 100-fold) than that reported in serum. The FS concentrations (ng/ml) were 203 +/- 42 (normal dominant), 185 +/- 17 (atretic cohort), 185 +/- 5 (IVF), and 250 +/- 14 (PCOS). There was no statistical difference between these mean values of FS. Further, there were no significant correlations between the follicular fluid concentrations of FS and the concentrations of oestradiol, progesterone, or androstenedione. These results indicate that human Graafian follicles, regardless of whether they are healthy or atretic, normal or PCOS, contain high steady-state concentrations of FS in the micro-environment. Collectively, these data fit with the hypothesis that major increases and decreases in the concentration of FS in the micro-environment may not play a key role in the mechanisms of selection, atresia, and PCOS in women. The possibility of regulation of intrinsic activin and inhibin activity through FS binding is discussed.

Case-Control Studies↗

PubMed1995

Interleukin-1 induces insulin-like growth factor binding protein-3 gene expression and protein production by Leydig cells.

Interleukin-1 (IL-1) is a potent inhibitor of Leydig cell function. IL-1 blocks human CG-induced cAMP and testosterone formation, as well as cytochrome P450 side-chain cleavage messenger RNA (mRNA) expression. IL-1 also decreases insulin-like growth factor-I (IGF-I) mRNA levels in Leydig cells. The effects of IGF-I are modified by IGF binding proteins (IGFBPs). In the present study, we evaluated the effects of IL-1 on IGFBP expression. Purified Leydig cells from adult rats were cultured with 0.1% heat-inactivated fetal bovine serum in Dulbecco's modified Eagles' medium/F12. Culture medium was changed to serum-free Dulbecco's modified Eagles' medium/F12 after 24 h and IL-1 beta (0.1-10 ng/ml) was added. Treatment of Leydig cells with IL-1 beta (10 ng/ml) for 2, 4, and 6 h resulted in a progressive induction of IGFBP-3 expression without affecting IGFBP-2 or IGFBP-4 mRNA levels. IL-1 beta in concentrations of 0.1, 1, and 10 ng/ml caused a 1.5-, 4-, and 6.5-fold induction of IGFBP-3 expression, respectively, whereas IGF-I mRNA levels were decreased in a dose-dependent manner. IL-1 beta increased the average transcription rate of IGFBP-3 by 3.3-fold. The t1/2 for IGFBP-3 mRNA was 2.07 h and was not affected by the treatment with IL-1 beta (2.21 h). The immunoblot of cell-conditioned media showed that the basal level of IGFBP-3 protein was low and IL-1 beta caused a dose-dependent increase in the production of IGFBP-3. These results indicate that IL-1 beta increases IGFBP-3 levels by increasing the rate of transcription rather than by changing the stability of IGFBP-3 mRNA. The addition of cycloheximide markedly inhibited IL-1 beta-induced IGFBP-3 mRNA levels. However, IL-1 beta was able to induce IGFBP-3 mRNA levels even in the presence of cycloheximide. This suggests that de novo protein synthesis may not be required for induction of IGFBP-3 mRNA by IL-1 beta. In conclusion, IL-1 beta inhibits IGF-I but increases IGFBP-3 expression in Leydig cells, and this may contribute to the inhibitory effects of IL-1 beta on Leydig cell steroidogenesis.

Animals↗

PubMed1994

Hormonal regulation of insulin-like growth factor II and insulin-like growth factor binding protein expression by breast cancer cells in vivo: evidence for stromal epithelial interactions.

Insulin-like growth factors (IGFs) I and II are potent mitogens for breast cancer cells. Their proliferative activity is likely to be influenced by their binding proteins (IGFBPs), a family of newly identified proteins. We report here on the in vivo hormonal regulation of mRNAs for IGF-II and IGFBPs in the N-nitrosomethylurea-induced rat mammary tumor, a well-established model of hormone-responsive mammary cancer. IGF-II mRNA levels tended to decrease in regressing tumors following ovariectomy, and they markedly increased upon reactivation of tumor growth with hormone repletion. Ovariectomy induced a drastic increase in IG-FBP-6 mRNA which was reversible with hormone repletion. Similar but more modest changes were observed with IGFBP-2 mRNA. In contrast, IGFBP-3 and IGFBP-4 mRNAs tended to decrease with ovariectomy and increase with hormone repletion. These latter effects, however, were modest, variable, and not statistically significant. In situ hybridization analysis revealed that IGF-II, IGFBP-5, and IGFBP-6 mRNAs were localized in the stromal component of the tumor, whereas IGFBP-2 mRNA was expressed by epithelial cells. We conclude that hormonal regulation of IGFBP expression is heterogeneous, thus suggesting divergent biological functions for these peptides. Our data also emphasize the importance of potential stromal-epithelial interactions in the control of breast cancer cell proliferation by IGFs.

Animals↗

PubMed1994

Follicle-stimulating hormone regulation of AP-1: inhibition of c-jun and stimulation of jun-B gene transcription in the rat Sertoli cell.

The proto-oncogenes c-fos and c-jun and the related jun-B encode the components of transcription factor, AP-1, a heterodimeric DNA-binding protein that mediates hormone and growth factor-regulated gene expression. In the rat Sertoli cell, FSH rapidly inhibited c-jun gene expression while it stimulated c-fos and jun-B as well as the expression of the more slowly responding, tissue plasminogen activator (tPA) and inhibin alpha-subunit. These early effects of FSH were not inhibited by cycloheximide. Nuclear run-off analyses demonstrated that the FSH-dependent decline in c-jun and increases in c-fos, jun-B, tPA and inhibin alpha-subunit mRNAs were regulated at the transcriptional level. The rates of degradation of c-fos, c-jun and jun-B mRNAs were unaffected by FSH while tPA and inhibin alpha-subunit mRNAs were stabilized. After 8 h of FSH treatment, the transcription of all five genes returned to basal rates. These data demonstrate immediate-early regulation by FSH of the expression of genes encoding components of the transcription factor, AP-1.

Animals↗

PubMed1994

Porcine ovarian granulosa cells secrete insulin-like growth factor-binding proteins-4 and -5 and express their messenger ribonucleic acids: regulation by follicle-stimulating hormone and insulin-like growth factor-1.

Using ligand blotting, Western immunoblotting, and Northern analysis, we have characterized the insulin-like growth factor (IGF)-binding proteins (IGFBPs) produced by cultures of porcine granulosa cells. Ligand blot analysis of conditioned medium from untreated cultures of moderately differentiated granulosa cells (MDGCs; from 4-6-mm follicles) revealed mainly IGF-binding activity associated with IGFBP-2 (34 kDa) and IGFBP-3 (40/44-kDa doublet), which have previously been identified and characterized. In addition, these cultures secreted 30- and 22-kDa forms under some circumstances. The identification and regulation of these IGFBPs of lower molecular mass were the focus of the current studies. Treatment of these MDGCs with IGF-I dramatically stimulated the production (to a detectable level) of a 30-kDa IGFBP that was identified by immunoblotting with antiserum to IGFBP-5 but not antisera to IGFBP-1, -2, -3, -4, or -6. Production of IGFBP-5 was attenuated by concurrent treatment with FSH. IGFBP-5 mRNA in these cultures was correspondingly stimulated by IGF-I but unaffected by FSH. FSH increased the level of a minor 22-kDa IGFBP. Messenger RNAs for IGFBP-1, -4, and -6 were also examined but only IGFBP-4 mRNA was detectable, suggesting that the 22-kDa band was IGFBP-4. These results were compared to those in cultures of immature granulosa cells from 1-3-mm follicles, in which 22- and 30-kDa IGFBPs were readily detectable. An antiserum to IGFBP-4 precipitated the 22- and 30-kDa bands whereas deglycosylation shifted the 30-kDa IGFBP to 22 kDa, suggesting that both these bands represent glycosylation variants of IGFBP-4.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Insulin-like growth factor binding proteins show distinct patterns of expression in the rat uterus.

An intrinsic insulin-like growth factor (IGF) system, complete with IGF ligands, receptors, and biological responses, is present in the rat uterus, where it is thought to regulate uterine homeostasis by autocrine/paracrine mechanisms. It is known that IGF binding proteins (IGFBP) modulate IGF-I and IGF-II action, but very little information is available concerning their cellular localization in the uterus. Therefore, we have employed in situ hybridization to localize IGFBP-1, -2, -3, -4, -5, and -6 mRNAs in the adult rat uterus during the estrous cycle. IGFBP-1 was undetectable in all uteri examined. IGFBP-2 mRNA was localized only in the luminal epithelium of the endometrium. It was abundant during proestrus (P1000 h, P2000 h) and early estrus (E0200 h), but was relatively low at other stages of the cycle. IGFBP-3 mRNA was localized to the stroma cells juxtaposed to the endometrium. A weak signal was detected on estrus morning (E0200 h, E1000 h), but high levels of IGFBP-3 mRNA were observed in the stroma cells on Day 12 of pregnancy. IGFBP-4 mRNA was localized only in the luminal epithelium of the endometrium. It was moderately abundant at diestrus I and II, but the signal was very low or absent at other times in the cycle. IGFBP-5 mRNA was localized in the circular and longitudinal muscle layers of the myometrium. The IGFBP-5 hybridization signal was maximal at diestrus, weak on proestrus, and moderate during estrus. IGFBP-6 mRNA was also expressed in the myometrium. The signal was strong on estrus morning (E0200 h and E1000 h) and low or absent at other times in the cycle. These results provide the first direct evidence that the genes encoding the six IGFBP are expressed in a tissue-specific manner in the adult rat uterus. Equally important, the levels of the mRNA for each IGFBP appear to change throughout the estrous cycle, but not in a parallel fashion. These results support the hypothesis that inducible and tissue-specific expression of IGFBP-2 to -6 may be involved in modulating the activity of the IGF ligands during the proliferative and secretory phases of the uterine cycle.

Animals↗

PubMed1994

Extrapituitary actions of gonadotropin-releasing hormone: stimulation of insulin-like growth factor-binding protein-4 and atresia.

To understand how the intrinsic GnRH system functions in the ovary, we tested the effects of GnRH agonist (GnRH-a) on insulin-like growth factor-binding protein-4 (IGFBP-4) production, a novel marker of atresia. We also tested the ability of GnRH-a to stimulate atresia in vivo. When rat granulosa cells were cultured in defined medium for 2 days (controls), relatively large amounts of the 24,000 relative molecular mass IGFBP-4 accumulated in the medium. FSH (100 ng/ml) inhibited control IGFBP-4 protein levels and stimulated IGFBP-4 protease activity. GnRH-a increased (up to 4-fold) IGFBP-4 accumulation in the medium (ED50 = 1 x 10(-10) M), and the effect was blocked by a GnRH antagonist. Neither GnRH-a nor its antagonist had a detectable effect on protease activity. In coincubation experiments, GnRH-a effectively inhibited (ED50 = 3 x 10(-11) M) the FSH responses, and the effect of GnRH-a was blocked by GnRH antagonist. A 6-day time-course experiment showed that IGFBP-4 accumulation in control cultures remained constant for 2 and 4 days, after which it was undetectable. FSH (100 ng/ml) produced no measurable IGFBP-4 over the 6-day time course. The levels of IGFBP-4 increased markedly during the first 2 days of GnRH-a treatment, but were not significantly different from control levels on days 4 and 6. Similar results were obtained when cells were treated with FSH plus GnRH-a. Treating immature hypophysectomized estrogen-primed rats with GnRH-a in vivo caused a rapid and dramatic decrease (average, 60%) in the mitotic index of the granulosa cells of all preantral follicles (healthy and atretic) and increased pyknosis. These results demonstrate that 1) GnRH-a stimulates the expression of IGFBP-4 protein in rat granulosa cells in vitro; 2) GnRH-a abolishes the ability of FSH to inhibit IGFBP-4 expression and induce IGFBP-4 protease activity; and 3) GnRH-a stimulates atresia in preantral follicles in vivo. These results support the hypothesis that autocrine/paracrine secretion of ovarian GnRH might cause atresia by mechanisms involving increased IGFBP-4 synthesis.

Animals↗

PubMed1994

Insulin-like growth factor-binding protein-2: the effect of human chorionic gonadotropin on its gene regulation and protein secretion and its biological effects in rat Leydig cells.

Human CG (hCG) and insulin-like growth factor-I (IGF-I) have synergistic effects on Leydig cell function. Leydig cells express high affinity IGF-I receptors. The number of IGF-I receptors and IGF-I receptor mRNA levels can be up-regulated by hCG. The most abundant mRNA species of the IGF binding proteins (IGFBPs) in rat Leydig cells is IGFBP-2. In the present study, we investigated the effect of hCG on IGFBP-2 transcription, mRNA accumulation, and protein production/secretion. Biological effects of IGFBP-2 on Leydig cells were also examined. Rat Leydig cells were purified from testes using centrifugal elutriation followed by Percoll gradient centrifugation. Cells were cultured for 24 h and then treated with or without hCG (10 ng/ml) for 6 h. The expression of IGFBP-2 mRNA was decreased by hCG in a dose-dependent manner, and at a concentration of 10 ng/ml the expression of IGFBP-2 mRNA was reduced by 50%. As early as 2 h after the addition of hCG, there was a significant decrease in IGFBP-2 mRNA accumulation. To evaluate the mechanism(s) responsible for decreased IGFBP-2 gene expression by hCG, the effect of hCG on the rate of transcription and stability of the mRNA was determined. Human CG (10 ng/ml) reduced the IGFBP-2 transcription rate by 32%/h in comparison with the control, while the half-life (t1/2) of mRNA remained unaltered (hCG-treated cells, 0.58 h; control cells, 0.51 h). IGFBP-2 with a molecular size of 33 kilodaltons was detected as a major band in the Western ligand blot.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Molecular heterogeneity of follistatin, an activin-binding protein. Higher affinity of the carboxyl-terminal truncated forms for heparan sulfate proteoglycans on the ovarian granulosa cell.

Follistatin (FS), an activin-binding protein, is a monomer derived from two polypeptide core sequences of 315 (FS-315) and 288 (FS-288) amino acids originated from alternatively spliced mRNA. To define the structural heterogeneity of native FS, we purified six molecular forms of FS from porcine ovaries. Protein chemical analysis revealed that the structural differences among the six isoforms were caused by truncation of the carboxyl-terminal region and/or the presence of carbohydrate chains, resulting in the formation of FS-315, FS-288, and FS composed of 303 amino acids (FS-303) in various forms of glycosylation on the two potential Asn-linked glycosylation sites. The majority of FS isolated from porcine ovaries was FS-303, which may have been derived from FS-315 by proteolytic cleavage of the 12 COOH-terminal amino acids. All six molecular species have almost the same activin binding activity (Kd = 540-680 pM). By contrast, the COOH-terminal truncated form, FS-288, showed much higher affinity for the rat granulosa cell surface than FS-303, whereas FS-315 had no affinity. FS-288 bound to heparan sulfate-Sepharose CL-4B, but FS-315 did not, suggesting that the truncated forms of FS bind to heparan sulfate proteoglycans on the cell. COS cells transfected with the FS-288 DNA expressed the FS-288 protein, which adhered to the cell surface, but cells transfected with the FS-315 DNA secreted the expressed protein into the medium, which did not bind to the cell surface. In rat anterior pituitary culture, FS-288 (ED50 = 2 ng/ml) was more potent in suppressing follicle-stimulating hormone release than FS-303 (ED50 = 10 ng/ml) and FS-315 (ED50 = 20 ng/ml). These results suggest that cell-associated FS traps activin more tightly in the matrix, thereby more effectively blocking the activity of activin on heparan sulfate proteoglycans of the cell surface and that cell-associated FS plays an important role in controlling the various actions of activin in a paracrine or autocrine manner.

Activins↗

PubMed1993

Differential control of activin, inhibin and follistatin proteins in cultured rat granulosa cells.

Follistatin, activin and inhibin proteins are produced by granulosa cells, but the mechanisms controlling their production remain unclear. Here, we examined how the protein kinase A (PKA) and protein kinase C (PKC) pathways act and interact to regulate production of these proteins. Granulosa cells from immature rats were cultured with activators of the PKA pathway (100 ng/ml FSH, 10 microM forskolin) and/or activators of the PKC pathway (100 nM GnRH agonist, 100nM 2-0-tetradecanoyl-phorbol-13-acetate, TPA). Conditioned media were assayed for inhibin and activin by ligand blotting using recombinant human 125I-follistatin and for follistatin by double ligand blotting using cold activin plus 125I-follistatin. FSH and forskolin stimulated inhibin but not activin production. In contrast, GnRH and TPA stimulated activin, and to a lesser degree, inhibin production; significantly, this is the first demonstration of activin dimer production by granulosa cells. Activators of the PKA pathway antagonized the actions of PKC effectors and vice versa. All agents increased follistatin protein production, and the PKA and PKC activators interacted to generate further increases in follistatin production. These results show that the FSH-PKA signalling pathway favors formation of alpha beta inhibin dimers while the GnRH-PKC pathway favors formation of beta-subunit activin dimers. Both pathways act to increase follistatin protein production.

Activins↗

PubMed1993

Structural characterization of the rat insulin-like growth factor binding protein-6 gene.

Recently a family of six distinct insulin-like growth factor binding proteins (IGFBPs) have been identified and the gene structures of the first five (IGFBP-1, -2, -3, -4 and -5) characterized. We now isolated the IGFBP-6 gene from a rat genomic library and determined its organization as well as the DNA sequence at the 5' flanking region of the gene. The rat IGFBP-6 gene spans 5.1 kb of the genomic sequence and contains four exons interrupted by three introns of approximately 2.4, 0.2 and 1.2 kb in length, respectively. Primer extension analysis and ribonuclease protection assay using RNA from rat lung tissues demonstrated two transcriptional start sites located at 85 and 82 nucleotides upstream of the ATG translational initiation codon. The promoter region of the rat IGFBP-6 gene is devoid of a TATA or a CAAT consensus sequence motif, but putative regulatory cis elements, including a Sp1, an estrogen receptor binding site and a retinoic acid responsive element, are present in the promoter region.

Amino Acid Sequence↗

PubMed1993

Cloning of the rat insulin- like growth factor binding protein-5 gene and DNA sequence analysis of its promoter region.

To understand the regulation of the insulin-like growth factor binding protein-5 (IGFBP-5) gene expression, we have cloned the IGFBP-5 gene from rat genomic libraries and determined its genomic organization as well as the DNA sequence at the 5' flanking region of the gene. The rat IGFBP-5 gene spans at least 17 kilobases (kb) of the genome and contains 4 exons interrupted by 3 introns of approximately 10, 0.6 and 0.7 kb, respectively. Southern blot analysis of the rat chromosome DNA revealed a single copy gene for IGFBP-5 in the haploid genome. Primer extension experiments demonstrated a single transcriptional start site located at 772 nucleotides 5' of the ATG translational start codon. In addition to a TATA box and a CAAT box, multiple putative cis-regulatory elements, including an AP-1, an AP-2 and a binding site for progesterone receptor are present in the promoter region. This finding suggests that the IGFBP-5 gene is one of those that are expressed in a tissue-specific manner.

Amino Acid Sequence↗

PubMed1993

Structure of the rat insulin-like growth factor binding protein-4 gene.

A family of six distinct insulin-like growth factor binding proteins (IGFBPs) have been isolated and their cDNA sequences characterized from rat and human species. Recently, the gene structures of the first three IGFBPs (IGFBP-1, -2 and -3) have also been determined. We now report the isolation of the rat IGFBP-4 gene and its genomic organization, as well as the DNA sequence of the promoter region. Rat IGFBP-4 gene spans at least 12 kilobases (kb) of the genomic sequence. It consists of four exons separated by three introns with approximate size of 6.4, 0.6 and 2.7 kb. A single transcriptional start site is located at 249 nucleotides upstream of the translational initiation ATG codon. The rat IGFBP-4 gene possesses a typical TATA box and a CAAT box, as well as multiple potential cis elements, including three cAMP responsive elements, three AP-1 binding sites and one progesterone receptor binding site in the 5' flanking region. The presence of these potential cis elements supports the tissue-specific expression of the IGFBP-4 gene.

Amino Acid Sequence↗

PubMed1993

Structural and functional characterization of the rat follistatin (activin-binding protein) gene promoter.

Follistatin was originally identified as a specific inhibitor of follicle stimulating hormone secretion and later characterized as a binding protein for activin. Since activin regulates hormone secretion and cell differentiation, the importance of understanding the mechanisms regulating the synthesis of its binding protein, follistatin, is evident. To study the regulation of follistatin gene expression, we first determined the transcription start site (cap site) of the rat follistatin gene using primer extension and ribonuclease protection assay. Our results led to the identification of multiple cap sites located at three different positions of the promoter. DNA sequence analysis revealed that each cap site was located at approximately 30 nucleotide (nt) downstream of three distinct TATA-like sequences. In primary cultures of rat granulosa cells, transfection studies using 5'-flanking regions of follistatin gene fused to the chloramphenicol acetyltransferase (CAT) reporter gene revealed the presence of two DNA segments that act to suppress basal transcriptional activity. The promoter activity of the CAT construct containing 2.6 kilo base pairs (kb) of 5'-flanking region was induced 2.5-fold above basal activity by forskolin (10 microM), and 1.6-fold by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM). Co-treatment with forskolin and TPA resulted in a 6.4-fold induction in its promoter activity, suggesting that two distinct signal transduction pathways, the cAMP-dependent protein kinase-A pathway and diacylglycerol-dependent protein kinase-C pathway, act coordinately to modulate follistatin gene transcription. Experiments using a series of 5'-flanking region deletion constructs located the regulatory regions responsive to these two pharmacological agents at nt -312 to -32 and -35 to +139.

Activins↗