Biomedical subjects

O Ritvos

Publications and source records attributed to O Ritvos.

At least 37 records · Page 2Linked to original sources

PubMed1998

Activin and bone morphogenetic protein (BMP) signalling during tooth development.

Activins and bone morphogenetic proteins (BMPs) belong to the transforming growth factor-beta (TGF-beta) superfamily of structurally related cytokines. Results of recent in vivo and in vitro studies suggest that activin A, several BMPs, and their cell surface receptors may participate in tooth development. In addition, follistatin an extracellular protein that may interact with activin and BMP signaling, appears to be involved. This review focuses on recent advances in relation to the roles of activin/BMP receptors, their ligands and extracellular modifiers during tooth development.

Activin Receptors↗

PubMed1997

The activin-binding protein follistatin is expressed in developing murine molar and induces odontoblast-like cell differentiation in vitro.

It has recently been shown that mice deficient in activin-beta A subunits and follistatin exhibit major defects in dentition. To increase understanding of the roles played by these molecules during tooth development, we determined the temporospatial expression of activin-beta A subunit and follistatin messenger RNA and their corresponding proteins in developing murine molars (between day E 14 and 2 days after birth). The effects of recombinant human activin A and its binding protein follistatin on odontoblast differentiation were also studied in cultures of dental papillae (DP) isolated from the mandibular first molars of E-17-day mice. In situ hybridization indicated that transcripts for activin-beta A subunit were abundant in pre-odontoblasts at the tips of forming cusps prior to odontoblast terminal differentiation, and transcripts for follistatin in overlying inner enamel epithelial cells (pre-ameloblasts). Pre-odontoblasts were also weakly immunoreactive in relation to activin-beta A subunit, pre-ameloblasts in relation to follistatin. When follistatin was added at different concentrations to a DP culture model (2-14 nmol/DP) together with heparin at constant concentration, differentiation of odontoblast-like cells was induced, as evidenced by polarization and deposition of extracellular matrix in vitro, to extents depending on the follistatin concentration. In contrast, the addition of activin A (2 nmol/DP) had no effect on the differentiation parameters studied. These findings suggest that the activin-follistatin system regulates odontoblast differentiation during tooth development. In particular, we suggest that binding of endogenous activin A by follistatin may allow odontoblast terminal differentiation to occur.

Activins↗

PubMed1997

Regulation of prostaglandin F2 alpha receptor expression in cultured human granulosa-luteal cells.

PGF2 alpha is a metabolite of arachidonic acid that triggers regression of the corpus luteum. Recent animal studies have indicated that PGF2 alpha (FP) receptor messenger ribonucleic acid (mRNA) is expressed in the corpus luteum. To understand the regulation of the FP receptor in the ovary we have cloned a partial complementary DNA (cDNA) sequence of the FP receptor from human granulosa cells obtained from women undergoing in vitro fertilization. The sequence of this cDNA is identical to the previously reported FP receptor sequences obtained from human uterine and placental cDNA libraries. Low levels of the FP receptor mRNA were observed in freshly isolated granulosa cells or in cultured granulosa-luteal (GL) cells, as detected by reverse transcriptase-PCR. hCG and 8-bromo-cAMP increased the steady state levels of the FP receptor mRNAs after incubation for 24-48 h, as detected by Northern blot hybridization. The stimulatory effect of hCG was concentration and culture stage dependent. Further, hCG and 8-bromo-cAMP increased binding of radiolabeled PGF2 alpha to intact GL cells. In contrast, phorbol 12-myristate 13-acetate inhibited basal as well as hCG- and 8-bromo-cAMP-induced FP receptor mRNA expression and binding of the radiolabeled ligand. In summary, hCG, 8-bromo-cAMP, and phorbol 12-myristate 13-acetate modulate the expression of the FP receptor in human GL cells, which may represent a mechanism to regulate the responsiveness of the ovary to PGF2 alpha.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Differential hormonal regulation of vascular endothelial growth factors VEGF, VEGF-B, and VEGF-C messenger ribonucleic acid levels in cultured human granulosa-luteal cells.

The development of ovarian follicles and subsequent corpus luteum formation is accompanied by very active angiogenesis. Ovarian granulosa cells produce vascular endothelial growth factor (VEGF), which is a potent endothelial cell mitogen and an angiogenic agent. The complementary DNAs of two other factors structurally related to VEGF, namely VEGF-B and VEGF-C, were recently cloned, but little is known of their regulation in the ovary. We first studied the expression of the messenger RNAs (mRNAs) of the three VEGF isotypes in freshly isolated human granulosa-luteal (GL) cells obtained at oocyte retrieval for in vitro fertilization. The hormonal regulation of these mRNAs was subsequently studied in primary cultures of human GL cells. Analysis of cultured GL cell RNA by reverse transcription-PCR revealed that these cells express the alternatively spliced transcripts representing 121-, 145-, and 165-amino acid VEGF isoforms. Northern blot hybridization analyses indicated that transcripts of 4.5 and 3.7 kilobases for VEGF, and 1.4 and 2.4 kilobases for VEGF-B and VEGF-C, respectively, are expressed in human GL cells. The basal VEGF mRNA levels declined steadily, whereas VEGF-B mRNA levels were rather invariant over a 10-day culture period of GL cells. In contrast, VEGF-C mRNA levels increased toward the end of culture. For studying the hormonal regulation of VEGF isotype mRNAs, GL cells were treated with hCG, recombinant human FSH, PGE2, as well as 8-bromo-cAMP and 12-O-tetradecanoylphorbol 13-acetate, which activate protein kinase A- and protein kinase C-dependent signaling pathways, respectively. All test agents stimulated the expression of VEGF mRNA levels in a concentration-dependent manner. Time-course studies indicated that all treatments induced VEGF mRNA levels as early as incubation for 2 h, and the effect was sustained up to 48 h. VEGF-B mRNA levels were not regulated by any of the test agents. However, we found that hCG and 8-bromo-cAMP decreased VEGF-C mRNA levels with a maximal response observed at 24 and 48 h after cellular treatment. We conclude that the mRNAs of VEGF, VEGF-B, and VEGF-C are expressed in human GL cells and that their mRNA steady state levels are regulated in cultured human GL cells in an isotype-specific manner. The differential regulation of VEGF, VEGF-B, and VEGF-C in human GL cells suggests that distinct VEGF isotypes may play different roles during the vascularization of the human ovarian follicle and corpus luteum.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1997

Induction of cyclooxygenase-2 and prostaglandin F2alpha receptor expression by interleukin-1beta in cultured human granulosa-luteal cells.

Prostanoids are important regulators of ovarian function, especially during ovulation and luteolysis. Cyclooxygenase (Cox) is the rate-limiting enzyme in conversion of arachidonic acid to prostanoids. We have examined the expression and regulation of the inducible Cox isoform (Cox-2) and of the receptor for PGF2alpha (FP) in human granulosa cells obtained from women undergoing oocyte retrieval for in vitro fertilization. Freshly isolated granulosa cells express Cox-2 and FP receptor messenger RNAs (mRNAs). FP receptor mRNA is also expressed in cultured human granulosa-luteal (GL) cells, but Cox-2 transcripts are expressed only upon induction. Interleukin-1beta (IL-1beta) elevated Cox-2 mRNA steady state levels in a concentration-dependent manner, and kinetic studies showed that Cox-2 mRNA levels were already induced at the 2 h point and returned to the basal level after incubation for 24 h. The protein synthesis inhibitor, cycloheximide, induced Cox-2 mRNA expression and potentiated the effect of IL-1beta. Degradation of Cox-2 mRNA was inhibited by IL-1beta, which suggests regulation at the posttranscriptional level. IL-1beta also induced the expression of Cox-2 protein, as detected by immunofluorescence staining using Cox-2-specific polyclonal antibodies. Further, IL-1beta-induced synthesis of prostanoids was blocked by a Cox-2-specific inhibitor, NS-398. In addition, hCG induced Cox-2 mRNA expression and potentiated the effect of IL-1beta. However, in contrast to the rapid and transient effect of IL-1beta on Cox-2 mRNA, the effect of hCG followed slower kinetics. We have previously shown that hCG induces expression of human FP receptor mRNA in cultured human GL cells. We now show that IL-1beta induces FP receptor mRNA in a time- and concentration-dependent manner. Our data suggest that Cox-2 and FP receptor are coexpressed in freshly isolated human granulosa cells and that their expression is up-regulated by IL-1beta and hCG in cultured human GL cells.

Cells, Cultured↗

PubMed1996

Differential regulation of inhibin/activin alpha- and beta A-subunit and follistin mRNAs by cyclic AMP and phorbol ester in cultured human granulosa-luteal cells.

Granulosa cell-derived inhibin A (a dimer of alpha- and beta A-subunits), activin A (a homodimer of beta A-subunits) and the activin-binding protein follistatin are important regulators of human ovarian steroidogenesis. We here studied how 8-bromo-cAMP (8br-cAMP), a protein kinase A activator, and 12-O-tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, affect the steady-state levels of alpha- and beta A-subunit and follistatin mRNAs in cultured human granulosa-luteal cells. 8br-cAMP induced alpha- and beta A-subunit and follistatin steady-state mRNA levels in a time- and concentration-dependent manner. The levels of alpha-subunit mRNAs were stimulated by 8br-cAMP in a sustained manner with a maximal induction seen at the time points 24 and 48 h. By contrast, beta A-subunit and follistatin mRNA levels were rapidly and transiently induced by 8br-cAMP with maximal effects observed at 3 h and 8 h, respectively. TPA did not affect basal alpha-subunit mRNA levels but it rapidly induced beta A-subunit mRNAs at 3 h and the stimulation was still evident at 48 h. TPA induced follistatin mRNA levels with kinetics similar to 8br-cAMP but to a lesser extent. Moreover, 8br-cAMP and TPA stimulated beta A-subunit and follistatin mRNA levels synergistically at 3 h. By contrast, TPA had a potent inhibitory effect on 8br-cAMP- and hCG-induced alpha-subunit levels. Neither 8br-cAMP nor TPA regulated inhibin/activin beta B-subunit mRNA levels. Taken together the activation of protein kinase-A and -C by 8br-cAMP and TPA, respectively, lead to clearly differential responses in the steady-state levels of inhibin activin alpha- and beta A-subunit and follistatin mRNAs. These results suggest that the inhibin A vs. activin A ratio as well as follistatin levels are regulated by multiple second-messenger pathways in the human ovary.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Prostaglandin E2 induces inhibin alpha- and beta A-subunit mRNA and secretion of dimeric inhibin A in cultured human granulosa-luteal cells.

Prostaglandins (PG) E2 and F2 alpha are produced by the human corpus luteum in significant amounts and they are known to exert local luteotrophic and luteolytic effects respectively. The present study determined how PGE2 and PGF2 alpha are involved in the regulation of inhibin A, a granulosa cell-derived glycoprotein hormone that has an endocrine inhibitory effect on hypophyseal gonadotrophin production and a local stimulatory action on thecal cell androgen secretion. The effects of PGE2 and PGF2 alpha on inhibin alpha- and beta A-subunit mRNAs levels and on the secretion of dimeric inhibin A were studied in cultures of human granulosa-luteal cells obtained from patients undergoing an in-vitro fertilization programme. We found that PGE2 induced mRNA of inhibin alpha- and beta A-subunits in a time- and concentration dependent manner, whereas PGF2 alpha had no clear effect. In contrast to the rapidly and transiently induced beta A-subunit mRNA, alpha-subunit mRNA accumulation was stimulated with slower kinetics by PGE2 in a protein synthesis-dependent manner; this was also observed for the mRNA of the cytochrome P450scc enzyme. In the medium of cells cultured for 24 h with PGE2 (1 x 10(-6) M), 1.3-1.6-fold increase (P < 0.05) above basal values in secreted dimeric inhibin A was observed. The results suggest that PGE2 is an important inducer of inhibin A production in granulosa cells and that a local action of PGE2 may partially explain the increase in circulating inhibin A observed during the midluteal phase of the human menstrual cycle.

Cells, Cultured↗

PubMed1996

Identification of ovarian granulosa cells as a novel site of expression for bone morphogenetic protein-3 (BMP-3/osteogenin) and regulation of BMP-3 messenger ribonucleic acids by chorionic gonadotropin in cultured human granulosa-luteal cells.

Bone morphogenetic proteins (BMP) belong structurally to the transforming growth factor-beta superfamily comprising several growth and differentiation factors such as inhibin, activin, and Müllerian inhibitory factor that regulate ovarian function. We studied here the potential expression of BMP-2, -3, and -4 messenger RNAs (mRNAs) in isolated human granulosa cells obtained at oocyte retrieval for in vitro fertilization. Freshly isolated granulosa cells were found to express BMP-3 (also known as osteogenin) mRNAs but not those of BMP-2 or -4. The BMP-3 transcripts were detected with RT-PCR amplification followed by Southern blot hybridization, as well as by Northern and dot blot hybridization analyses. To investigate whether BMP-3 mRNAs are hormonally regulated, cultures of human granulosa-luteal (GL) cells were treated with different concentrations of purified human chorionic gonadotropin (hCG) at varying stages of culture. hCG decreased BMP-3 mRNA levels from the first day of the culture up to day 5. Time-dependence studies showed that a clear decrease in BMP-3 mRNA levels was evident at 24 h after hCG treatment, and that the effect of hCG was concentration dependent with 3 ng/mL hCG decreasing significantly (P < 0.05) BMP-3 mRNA levels. Furthermore, the cAMP analog, 8-bromo-cAMP (8-Br-cAMP), which activates protein kinase-A, and 12-0-tetradecanoylphorbol 13-acetate, an activator of protein kinase-C, both markedly decreased BMP-3 mRNA levels in an 8-h treatment. We conclude that: 1) BMP-3 mRNAs are expressed in human granulosa cells; 2) their steady state levels are hormonally regulated in cultured human GL cells as evidenced by the ability of hCG to markedly decrease BMP-3 transcript levels; and (3) that activation of both protein kinase-A-and protein kinase-C-mediated signaling pathways also results in a decrease in BMP-3 mRNA levels in GL cells. We suggest that BMP-3, like several other members of the transforming growth factor-beta superfamily, is a potential local regulator of female gonadal function.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1996

Transforming growth factor-beta 1 and -beta 2 induce inhibin and activin beta B-subunit messenger ribonucleic acid levels in cultured human granulosa-luteal cells.

OBJECTIVE: To examine the effect of transforming growth factor-beta (TGF-beta) on inhibin and activin subunit messenger ribonucleic acids. DESIGN: Human granulosa-luteal cell culture model. SETTING: Granulosa cells were obtained from women undergoing an IVF program in a private IVF clinic. PATIENTS: Regularly menstruating women undergoing oocyte retrieval for IVF because of either tubal obstruction or infertility of the spouse. INTERVENTIONS: For each experiment, cells of two to four patients were pooled, enzymatically dispersed, separated from red blood cells by centrifugation through Ficoll-Paque and cultured in vitro in the presence of TGF-beta 1 or TGF-beta 2 and/or hCG whereafter cellular RNA was extracted for Northern or dot blot filter hybridization with inhibin alpha-, beta A, and beta B-subunit complementary DNA probes. RESULTS: Both TGF-beta 1 and TGF-beta 2 induced the expression of a 4.8-kb inhibin and activin beta B-subunit messenger (mRNA) transcript in a time- and dose-dependent manner but had no effect on alpha- or beta A-subunit mRNA levels. Human chorionic gonadotropin alone did not affect beta B-subunit mRNA levels, but when administered together with TGF-beta s, it prevented the induction of beta B-subunit mRNAs. CONCLUSIONS: Our results suggest that in human ovary, granulosa, or thecal cell-derived TGF-beta 1 or -beta 2 may eventually locally modulate in a paracrine or autocrine manner the relative expression levels of inhibin and activin subunits favoring the formation of the inhibin and activin dimers containing the beta B-subunit. The effect of TGF-beta is clearly different from that of gonadotropins, which potently induce the alpha- and beta A-subunit mRNAs, indicating that distinct components of the human ovarian inhibin and activin system are regulated differentially by endocrine and local factors.

Activins↗

PubMed1995

Activin disrupts epithelial branching morphogenesis in developing glandular organs of the mouse.

We report that activin profoundly alters epithelial branching morphogenesis of embryonic mouse salivary gland, pancreas and kidney rudiments in culture, indicating that it may play a role as a morphogen during mammalian organogenesis. In developing pancreas and salivary gland rudiments, activin causes severe disruption of normal lobulation patterns of the epithelium whereas follistatin, an activin-binding protein, counteracts the effect of activin. In the kidney, activin delays branching of the ureter bud and reduces the number of secondary branches. TGF-beta induces a pattern of aberrant branching in the ureter bud derived epithelium distinct from that seen for activin. Reverse-transcriptase polymerase chain reaction, Northern hybridization and in situ hybridization analyses indicate that these developing tissues express the mRNA transcripts for activin subunits, follistatin or activin receptors. Our results are suggestive of a potential role for the activin-follistatin system as an intrinsic regulator of epithelial branching morphogenesis during mammalian organogenesis.

Activin Receptors↗

PubMed1995

Regulation of the activin-binding protein follistatin cultured human luteinizing granulosa cells: characterization of the effects of follicle stimulating hormone, prostaglandin E2, and different growth factors.

Regulation of the activin-binding protein follistatin (FS) by recombinant human (rh) FSH, prostaglandin E2 (PGE2), and several polypeptide growth factors was examined in cultures of human granulosa-luteal (GL) cells obtained from in-vitro fertilization patients at oocyte retrieval. Northern and dot blot hybridization analyses demonstrated that both rhFSH and PGE2 caused stimulatory effects on FS mRNA levels in a culture stage-, time-, and concentration-dependent manner. An 8-h stimulation with rhFSH (100 ng/ml) significantly increased FS mRNA levels on Days 5 and 7 of culture and PGE2 (10(-6)M) on Days 2, 4, and 7. The stimulatory effect of rhFSH and PGE2 on FS mRNA levels were rapid and transient. Maximal inductions occurred 8 h after stimulation, whereas weak or no stimulatory effects were seen at 24 or 48 h. PGF2 alpha did not affect FS mRNA levels at any time point studied. Treatment of the cells with the protein synthesis inhibitor cycloheximide prior to rhFSH stimulation did not inhibit the rapid induction of FS mRNAs, but it prevented the decline at 24 h. Both rhFSH and PGE2 clearly also increased the levels of secreted FS proteins are detected by immunoprecipitation studies with a specific antibody. The effects of the polypeptide growth factors epidermal growth factor (EGF); transforming growth factor beta 1 (TGF beta 1), and activin A on FS mRNA levels were also examined. TGF beta 1 and activin A had no effect on basal FS expression at any concentration or time point studied. An 8-h stimulation with EGF increased FS mRNA levels, but the effect was weaker than those caused by rhFSH and PGE2. We conclude that rhFSH and PGE2 induce FS mRNA and protein in human cultured GL cells. EGF is able to induce FS mRNA to a lesser extent than are rhFSH and PGE2, whereas PGF2 alpha, TGF beta 1, and activin A do not affect basal FS mRNA levels in human cultured GL cells. This study together with our previous report on the stimulatory effect of hCG on FS levels suggest that in the luteal phase of the human menstrual cycle, FS expression in granulosa cells is likely to be positively controlled by luteotropic factors such as gonadotropins and PGE2. Consequently, elevated FS levels may support the survival of the human CL since FS is known to prevent the antisteroidogenic effects of activin in human GL cells.

Activins↗

PubMed1995

Expression of c-kit ligand messenger ribonucleic acids in human ovaries and regulation of their steady state levels by gonadotropins in cultured granulosa-luteal cells.

The c-kit ligand (KL), a ligand for the c-kit protooncogene receptor tyrosine kinase, is an important regulator of germ cell development in rodent gonads. However, no information about the role of KL in the ovaries of women or higher primates has been available. We studied the expression of KL messenger RNA (mRNA) in human ovaries and the effect of purified hCG and recombinant human FSH (rhFSH) on KL mRNA steady state levels in cultures of human granulosa-luteal (GL) cells obtained at oocyte harvest for in vitro fertilization. KL complementary DNA was generated by reverse transcription-polymerase chain reaction from human ovarian tissue RNA. Two alternatively spliced KL transcripts encoding 248-amino acid (aa) and 220-aa membrane-associated KL proteins were observed in GL cells and ovarian tissue. In Northern blot analysis of human ovarian and GL cell RNA, a major transcript of approximately 6.0 kilobases was detected. Specific mRNA transcripts for KL were detected in dot blot filter hybridization analyses, and the steady state levels of these mRNAs were lowered in cultured GL cells by both gonadotropins in a distinct time- and concentration-dependent manner. The KL mRNA levels of untreated and hCG- or rhFSH-stimulated GL cells were determined at 2- to 3-day intervals between days 2-10 of culture. An 8-h treatment with hCG was shown to decrease KL mRNA levels on days 2, 3, 5, and 7 of culture, whereas rhFSH decreased KL mRNA levels on days 5 and 7 of culture. Time-course and concentration-dependence studies were performed on days 2-7 of culture. Both gonadotropins decreased KL mRNA levels as early as 2 h after treatment. The maximal response to hCG and rhFSH treatment was observed at 7-24 h. Concentration-dependence studies performed 8 or 24 h after treatment indicated that the maximal inhibition occurred with 10-100 ng/ml hCG and 100-300 ng/ml rhFSH. We conclude that 1) the KL transcripts encoding 248- and 220-aa transmembrane proteins are expressed in vivo in the human ovary and in cultured human GL cells; and 2) KL transcript levels are rapidly decreased by gonadotropins in a time- and concentration-dependent manner in cultured GL cells. Thus, KL expression is hormonally regulated in human granulosa cells, and this growth factor may control the function of the ovarian follicle during the human menstrual cycle.

Adult↗

PubMed1995

Regulation of inhibin/activin subunit messenger ribonucleic acids (mRNAs) by activin A and expression of activin receptor mRNAs in cultured human granulosa-luteal cells.

Recent studies have indicated that activin and inhibin may act as local regulators of cell growth and steroidogenesis in the human ovary. We studied the effect of recombinant human activin A and purified bovine inhibin A on the steady state messenger RNA (mRNA) levels of the inhibin/activin alpha-, beta A-, and beta B-subunits in cultured granulosa-luteal (GL) cells from preovulatory ovarian follicles of women undergoing in vitro fertilization. Activin A induced the expression of a 4.8-kilobase beta B-subunit mRNA transcript without affecting basal expression levels of the alpha- and beta A-subunit mRNAs. It stimulated beta B-subunit mRNA levels in a concentration- and time-dependent manner. Maximal stimulation of beta B-subunit mRNA levels was obtained with 30-100 ng/ml activin A. The level of beta B-subunit mRNAs increased significantly 8 h after stimulation, rising gradually thereafter to a maximum at 48 h. Inhibin A did not affect the mRNA levels of any inhibin/activin subunits, nor did it inhibit the effect of activin A. Recombinant human follistatin did not affect basal beta B-subunit mRNA levels, but it neutralized the effect of activin A. Although hCG induces inhibin/activin alpha- and beta A-subunit mRNA levels in human GL cells, it did not increase basal beta B-subunit levels. By contrast, it inhibited activin A-induced beta B-subunit mRNA levels. On the other hand, activin A decreased hCG-induced mRNA levels of the inhibin alpha-subunit and cytochrome P450 side-chain cleavage (P450scc) enzyme, an important rate-limiting enzyme in human GL cell progestin synthesis. Moreover, we observed by Northern blot analysis that cultured human GL cells as well as freshly isolated preovulatory granulosa cells express the specific mRNAs for all currently known serine/threonine kinase activin receptors, i.e. activin receptors I, IB, II, and IIB. Our results suggest that in GL cells, activin A may locally stimulate synthesis of the beta B-subunit in an autocrine or paracrine manner, and that in human ovary, regulation of the beta B-subunit differs from that of the alpha- and beta A-subunits.

Activin Receptors↗

PubMed1994

Expression of type II activin receptor genes during differentiation of human K562 cells and cDNA cloning of the human type IIB activin receptor.

Recent studies have indicated that activin A/erythroid differentiation factor is a physiologic hematopoietic growth and differentiation factor mainly for cells of the erythroid lineage. We studied the expression of the two type II activin receptor mRNAs during the differentiation of K562 erythroleukemic cells, which are known to be induced toward the erythroid lineage in response to activin or toward the megakaryoblastic lineage by phorbol myristate acetate (PMA). The cDNA of the human activin receptor type IIB (hActR-IIB) was cloned and sequenced from two RNA sources, the K562 cells and the human fetal brain, which is, of the tissues screened by Northern blot analysis, the most abundant source of ActR-IIB RNA. The cDNA encodes a predicted 512 amino acid protein containing an extracellular ligand binding domain, a hydrophobic transmembrane domain, and an intracellular serine/threonine kinase domain. The amino acid sequence is 99.2% and 98.4% homologous in the coding region to the previously described mouse and rat ActR-IIB2s, respectively, and 69% identical to the other human activin serine/threonine kinase receptor, hActR-II. The alternative splicing events in the juxtamembrane region previously reported for the respective mouse receptor were not observed during the processing of K562 cell and human fetal brain RNA. Northern analysis showed that the 10- and 2.5-kb transcripts of hActR-IIB are more abundantly expressed than the 6.0- and 3.0-kb transcripts of hActR-II in K562 cells. No changes in the steady-state levels of hActR-II and IIB mRNAs were detected upon differentiation of K562 cells by activin A or by PMA. Similarly, the receptor mRNA levels remained constant in HL-60 cells induced to either monocyte/macrophage or granulocyte-like cells by PMA or dimethyl sulfoxide, respectively. Thus, the mRNA expression levels of both receptors apparently do not correlate with the differentiation status of these cells.

Activin Receptors↗

PubMed1994

Activin-binding protein follistatin messenger ribonucleic acid and secreted protein levels are induced by chorionic gonadotropin in cultured human granulosa-luteal cells.

We studied the effect of hCG on follistatin (FS) messenger RNA (mRNA) steady state levels and protein secretion in cultures of human granulosa-luteal (GL) cells obtained at oocyte harvest for in vitro fertilization. Three different overlapping FS complementary DNA (cDNA) fragments were generated by reverse transcription-polymerase chain reaction from human GL cell RNA. Together, these fragments covered the open reading frame, which appeared to be identical in sequence to previously isolated human testis-derived cDNAs. An alternative splicing event at the 3'-end of the FS transcript previously shown to give rise to transcripts encoding 344 amino acid (aa) and 317-aa proteins was also observed. In Northern analysis of human GL cell RNA, a major 2.5-kilobase transcript and a minor 1.5-kilobase FS transcript were detected, and the steady state levels of both mRNAs were induced by an 8-h stimulation with hCG (30 ng/ml). Time and concentration dependence studies on the effect of hCG were performed with cells cultured for 6-8 days before hormone treatment. Time-course experiments indicated that hCG (30 ng/ml) markedly induces FS mRNA levels as early as 2 h after stimulation. The maximal response to hCG stimulation, about 9-fold (mean of five experiments) above basal levels, was observed at 6-8 h, and thereafter, only moderate or no induction of FS mRNA levels could be detected at 24 or 48 h. Concentration dependence studies performed 8 h after stimulation indicated that the maximal induction occurred with 30-100 ng/ml hCG, with an ED50 of about 3-10 ng/ml. When the cells were treated with the protein synthesis inhibitor cycloheximide (20 micrograms/ml) 20 min before stimulation of the cells with hCG, both basal and hCG-stimulated FS mRNA levels increased at 24 h, indicating stabilization of the transcripts. However, it did not affect the rapid induction of FS mRNA levels by hCG at 2 h. The decline in FS transcript levels in untreated and hCG-treated cells was studied by blocking the transcription with 5 microM actinomycin-D. The degradation rate of FS mRNA was increased in hCG-treated compared to control cells. To study whether the transiently induced FS mRNAs are translated to proteins in hCG-treated and untreated human GL cells, metabolic labeling and immunoprecipitation experiments were performed to detect secreted [35S]FS proteins with the specific anti-FS antiserum Rb 32.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1994

The tissue distribution of activin beta A- and beta B-subunit and follistatin messenger ribonucleic acids suggests multiple sites of action for the activin-follistatin system during human development.

Activins have potent effects on early morphogenetic events during amphibian embryogenesis but no evidence for their role during human development other than their expression in steroidogenic tissues has been reported. We previously showed the expression of the activin type II and IIB receptor mRNAs in several tissues of the mid-gestational human fetus with highest expression levels in developing neural, muscular and exocrine glandular organs. We now report that the mRNA transcripts for activin beta A- and beta B-subunits and for the activin-binding protein follistatin are found co-expressed in several of these extragonadal tissues. Their mRNAs were detected by Northern analyses using specific single-stranded 32P-labeled cDNA probes. In the nervous system, both activin beta A- and beta B-subunit transcripts were expressed in the cerebrum and spinal cord. Follistatin was abundantly expressed in the spinal cord whereas weaker signals where observed in the cerebrum and cerebellum. In the muscular system, beta A-subunit was abundantly expressed in the heart but to a lesser extent in the skeletal muscle while the opposite was observed for follistatin. Follistatin, and activin beta A- and beta B-subunit mRNAs were also detected in developing kidney, salivary gland, liver, and adrenal. The predominance of beta A-subunit mRNAs in the bone marrow and beta B-subunit mRNAs in the salivary gland suggests specific roles for activin A and B, respectively, in these tissues. No hybridization signal was detected for the inhibin alpha-subunit in non-steroidogenic tissues indicating that, in contrast to activins and follistatin, the effects of inhibins may be restricted to the gonads and adrenals which are known to express high levels of the alpha-subunit transcript. Taken together, our results suggest that the activin-follistatin system regulates the development of several organ systems in the mid-gestational human fetus.

Abortion, Legal↗

PubMed1994

Regulation of inhibin alpha- and beta A-subunit messenger ribonucleic acid levels by chorionic gonadotropin and recombinant follicle-stimulating hormone in cultured human granulosa-luteal cells.

We studied the effects of recombinant human FSH (rhFSH) and purified hCG on the steady state messenger ribonucleic acid (mRNA) levels of inhibin alpha- and beta A-subunits in cultured granulosa-luteal cells of preovulatory ovarian follicles obtained from women undergoing in vitro fertilization. Specific mRNA transcripts for the alpha- and beta A-subunits were detected in Northern and dot blot filter hybridization analyses, and the levels of these mRNAs were induced by rhFSH and hCG in a distinct concentration- and time-dependent manner. The basal and hCG-stimulated alpha-subunit mRNA levels were first determined at 2- to 3-day intervals over a 3- to 10-day culture period after the initiation of the cultures. Both the basal and hCG-stimulated alpha-subunit mRNA levels declined steadily during culture, but the maximal relative stimulatory effect of hCG was observed on day 7 of culture. All subsequent experiments, therefore, were performed on days 6-8 of culture. Both gonadotropins induced alpha-subunit mRNA levels with slower kinetics than those of the beta A-subunit. Varying between experiments, rhFSH and hCG increased the expression of the alpha-subunit with a maximal effect of 2.5- to 5.7-fold and 1.7- to 7.2-fold, respectively, above basal levels 24-48 h after stimulation. rhFSH and hCG induced beta A-subunit mRNA levels with 3.0- to 5.8-fold and 2.3- to 8.6-fold increases above basal levels, respectively, at 2 h; thereafter, only moderate or no stimulation of the beta A-subunit mRNA levels could be detected at 7-48 h. Treatment of the cells with the RNA synthesis inhibitor actinomycin-D prevented the induction of alpha-subunit mRNA levels by hCG, and no significant differences were detected in the stability of alpha-subunit mRNA transcripts in hCG-treated cells vs. untreated cultures. This indicates that hCG induces transcription of the alpha-subunit gene rather than maintains the levels of preexisting transcripts. As the kinetics of induction of alpha- and beta A-subunit mRNAs by gonadotropins were different, we examined how the inhibition of protein synthesis affects the induction of alpha- and beta A-subunit mRNAs by hCG. Cycloheximide had no effect on basal alpha-subunit mRNA levels at 2 or 24 h. However, it inhibited at 24 h the induction of the alpha-subunit by hCG.(ABSTRACT TRUNCATED AT 400 WORDS)

Blotting, Northern↗

PubMed1993

Inhibin/activin subunit mRNA expression in human granulosa-luteal cells.

We studied the expression of inhibin/activin subunit mRNAs in granulosa-luteal cells of preovulatory ovarian follicles obtained from women undergoing in vitro fertilization, and in corpus luteum tissue samples of early pregnancy. Northern analysis of granulosa-luteal cell and corpus luteum RNA with single-stranded cDNA or cRNA probes revealed an 1.6-kb mRNA for the alpha subunit and about 6.0-, 4.0-, 2.8-, and 1.7-kb transcripts for the beta A subunit. No clear hybridization signal for the beta B subunit could be detected. The relative expression levels of alpha and beta A subunit mRNAs were determined at 2-day intervals in granulosa-luteal cells cultured for 5 to 11 days. The levels of alpha subunit mRNAs declined steadily with increasing culture age, whereas those of beta A remained unchanged. Reverse transcription-polymerase chain reaction analysis with 35 amplification cycles confirmed the expression of alpha and beta A subunit mRNAs in cultured granulosa-luteal cells. The beta B transcripts were also weakly detectable by this sensitive assay. In situ hybridization of human early pregnancy corpus luteum revealed intense hybridization with the alpha cRNA probe and a weaker signal for the beta A subunit in the granulosa cell compartment. We conclude that: (1) the inhibin alpha and beta A subunits (and to a lesser extent beta B) are expressed in cultured human granulosa-luteal cells; (2) during extended culture periods the alpha/beta A mRNA expression ratio decreases; and that (3) the alpha and beta A subunit mRNA expression is observed in the granulosa cell compartment of early pregnancy corpora lutea.

Activins↗