Activin Beta Research Articles

Expression and function of activin beta A during mouse cardiac cushion tissue formation

Expression and function of activin beta A during mouse cardiac cushion tissue formation

Expression and secretion of inhibin and activin in normal and neoplastic uterine tissues. High levels of serum activin A in women with endometrial and cervical carcinoma.

Inhibins and activins are growth factors belonging to the transforming growth factor-beta) family and are known to influence cell proliferation and differentiation. Because transforming growth factor-beta is involved in physiological and tumoral changes of uterine tissues, the present study aimed to evaluate whether human normal and neoplastic endometrial and cervical epithelial cells express and secrete inhibin A, inhibin B, and activin A. To test this hypothesis, different approaches were used. By RT-PCR, the expression of specific messenger RNAs (mRNAs) for the inhibin alpha, activin betaA and betaB subunits, and activin receptor type II and type IIB was investigated: 1) in primary cultures of endometrial (stroma and epithelium) or cervical (epithelium) cells from healthy women; and 2) in specimens of endometrial or cervical carcinoma. To demonstrate a possible secretion of the proteins, dimeric inhibin A, inhibin B, and activin A were measured in culture medium of normal epithelial or stromal endometrial cells and in uterine washing fluid of healthy women or patients with endometrial adenocarcinoma. Levels of the proteins were also measured in serum of women with endometrial or cervical carcinoma. Cultured endometrial stromal or epithelial cells and epithelial cervical cells expressed inhibin alpha, activin betaA and betaB, and activin receptor type II and type IIB mRNAs. The same finding was obtained in specimens of endometrial or cervical carcinomas. Dimeric inhibin A, inhibin B, and activin A were measured in culture medium of both endometrial and cervical cells. In particular, resulting activin A levels were significantly higher in epithelial than in stromal cultured endometrial cells (P < 0.01). Dimeric proteins were also detected in the washing fluid of the uterine cavities of healthy women (controls) and with endometrial adenocarcinoma, in which higher activin A levels were found (P < 0.01 vs. controls). Women with endometrial carcinoma showed serum activin A levels significantly higher than healthy controls (P < 0.01), which significantly decreased after surgical removal of endometrial or cervical tumors (P < 0.01). The present study, for the first time, showed that inhibin A, inhibin B, and activin A, as well as activin receptors, are expressed in normal and neoplastic human uterine tissues. A secretion of activin A from tumoral cells into systemic circulation is suggested by the observation that the high levels in serum of patients with endometrial or cervical carcinoma decreased after the surgical removal of the tumor.

Inhibins, activins, and follistatins: expression of mRNAs and cellular localization in tissues from men with benign prostatic hyperplasia.

The transforming growth factor beta (TGF beta) superfamily of growth factors includes activins and inhibins, which have been shown to be present in the rat ventral prostate, and human prostate tumor cell lines, although their localization in benign prostatic hyperplasia (BPH) tissue is currently unknown. BPH tissues were obtained at surgery, and the mRNA expression for the inhibin alpha, beta A, beta B subunits, the putative activin beta C subunit, the activin type II receptor (ActRII), and the activin binding protein, follistatin, was determined by reverse transcription polymerase chain reaction (RT-PCR) and Southern blot analysis. Antibodies specific for alpha, beta A, beta B, activin A, and follistatin were used to determine the localization of these proteins in BPH tissue specimens. Southern blot analysis confirmed that mRNA for ActRII, beta C subunit, and follistatin was present in all biopsy samples assayed. However, alpha, beta A, and beta B subunit mRNA expression was variable between patient samples. Immunohistochemistry demonstrated the predominant localization of beta A, beta B, and activin A proteins to the epithelium of BPH tissues. No immunoreactivity for the inhibin alpha subunit was detected; follistatin immunoreactivity was localized to the fibroblastic stroma. The compartmentalization of activin subunit proteins to the epithelium, and of follistatin to the stroma, suggests that a paracrine interaction occurs between the activin ligands and follistatin-binding proteins in BPH tissue.

Overexpression of activin A inhibits growth, induces apoptosis, and suppresses tumorigenicity in an androgen-sensitive human prostate cancer cell line LNCaP

The effects of overexpression of activin A in the androgen-sensitive human prostate cancer cell line LNCaP were studied. A full-length cDNA of activin beta A coding region was inserted into a eukaryotic expression vector and transfected into the LNCaP cells. Overexpression of activin BA significantly inhibited growth of this cell line. An increased death rate was also noted in these activin-overproducing cells, which was believed to be due to apoptosis as manifested by morphological change, DNA laddering, and FAGS analysis. The expression of bcl-2 was suppressed and the expression of c-myc was stimulated in these cells. In addition, the efficiency of soft agar colony formation and the tumorigenicity in the nude mice were suppressed for the activin producing LNCaP cells.

Focal brain injury increases activin betaA mRNA expression in hippocampal neurons.

The role of activin in the mammalian brain is of increasing interest as evidence accumulates to suggest a number of different neural functions. Here, we report that activin betaA mRNA is transiently induced in dentate gyrus neurons after unilateral mechanical brain injury by saline injection. Expression is dependent on NMDA receptor activation since pretreatment with MK801 (5 mg/kg, i.p.) largely attenuates the signal. Induction also requires de novo protein synthesis, as cycloheximide (10 mg/kg, i.p.) pretreatment abolishes the expression of activin betaA mRNA 1 h after injury. These results show that activin betaA mRNA expression is regulated by excitatory activity induced by focal brain injury and suggests a possible neuroplastic role in the recovery from such injury.

Activins and activin receptors in cell growth.

Activin and inhibin, members of transforming growth factor-beta (TGFbeta) superfamily, have diverse and widespread effects within living organisms at many stages during growth and development. From the initial isolation of these growth factors based on their effects of FSH secretion, the study of these factors, as well as of the activin-binding protein follistatin, has progressed from the localization of the expression of the inhibin alpha subunit, activin betaA and betaB subunits, and activin receptors in the tissues of various organisms to the examination of activin and inhibin as autocrine and paracrine agents in cell proliferation and differentiation. The inhibitory effects on cell growth and differentiation that have been observed upon treatment of cells with activin suggest that further understanding of the bioactivity of this molecule and its characterization on a molecular level may aid in a more complete understanding of cell growth and differentiation. This minireview discusses the roles of activin, inhibin, and follistatin in the arenas of cell proliferation, differentiation, and embryogenesis, as well as the roles of these molecules in cancerous cells.

Human Activin βA Gene IDENTIFICATION OF NOVEL 5′: EXON, FUNCTIONAL PROMOTER, AND ENHANCERS

On the basis of cDNA cloning, primer extension, and transfection experiments, we identified a novel 5' exon of the human activin betaA subunit gene, and found its enhancer and promoter regions as well as multiple transcription start sites. A series of deletion and mutation analyses of the enhancer sequences defined the 45-base pair core region (DR-1 core) containing two short elements with similarity to AP-1 (12-O-tetradecanoylphorbol-13-acetate response element; TRE) and CREB/ATF (cyclic AMP response element; CRE) binding sites, both of which were necessary for full enhancer activity. Gel shift and antibody supershift assays using DR-1 core region revealed the formation of two specific DNA-protein complexes, one of which could be partially dissociated by a competing oligonucleotide containing a single copy of the consensus TRE, but the other of which contained neither CREB/ATF nor AP-1 as major components. Although 12-O-tetradecanoylphorbol-13-acetate and cAMP induced the activin enhancer/promoter-driven CAT activity, such drug induction was obscured when either the TRE- or CRE-like elements were mutated in the native promoter context. Our results demonstrate that the promoter and enhancer regions identified here are essential for maintaining the efficient promoter activity of the human activin betaA subunit gene.

Follistatin and activin in bone: expression and localization during endochondral bone development

The involvement of activin and follistatin, an activin-binding protein, in endochondral bone development was examined by sc implantation of demineralized bone matrix in rats. Immunoreactive follistatin was localized in proliferating chondrocytes and round osteoblasts, whereas it was not detected in hypertrophic chondrocytes and osteoblasts surrounding bone marrow. Western blot analysis also revealed that immunoreactive follistatin was higher during the initial stages of chondrogenesis (day 5) and osteogenesis (days 11 and 14) and lower during the conversion from cartilage to bone (day 9). These results suggest that follistatin is produced by proliferating cells, and the expression decreases with differentiation of the cells. Implants injected with follistatin on days 9 and 10 contained lower calcium levels on day 14 than those injected with rat albumin. Furthermore, the follistatin-injected implants were still mainly composed of cartilage, suggesting that the disappearance of follistatin is necessary for the conversion of cartilage to bone. In contrast, immunoreactive activin beta A (55-60 kDa) was continuously detected in implants on days 7-14. The content of C propeptide of type II procollagen was increased and cartilageous area was enlarged on day 7 by activin A injections on days 5 and 6, suggesting a chondrogenic effect of activin in the initial stage of cartilage formation. These results indicate that proliferating chondrocytes and round osteoblasts produce follistatin, and that the activity of activin is regulated by changes in the expression of follistatin at the stages of chondrogenesis and transition from cartilage to bone.

Follistatin and activin in bone: expression and localization during endochondral bone development.

The involvement of activin and follistatin, an activin-binding protein, in endochondral bone development was examined by sc implantation of demineralized bone matrix in rats. Immunoreactive follistatin was localized in proliferating chondrocytes and round osteoblasts, whereas it was not detected in hypertrophic chondrocytes and osteoblasts surrounding bone marrow. Western blot analysis also revealed that immunoreactive follistatin was higher during the initial stages of chondrogenesis (day 5) and osteogenesis (days 11 and 14) and lower during the conversion from cartilage to bone (day 9). These results suggest that follistatin is produced by proliferating cells, and the expression decreases with differentiation of the cells. Implants injected with follistatin on days 9 and 10 contained lower calcium levels on day 14 than those injected with rat albumin. Furthermore, the follistatin-injected implants were still mainly composed of cartilage, suggesting that the disappearance of follistatin is necessary for the conversion of cartilage to bone. In contrast, immunoreactive activin beta A (55-60 kDa) was continuously detected in implants on days 7-14. The content of C propeptide of type II procollagen was increased and cartilageous area was enlarged on day 7 by activin A injections on days 5 and 6, suggesting a chondrogenic effect of activin in the initial stage of cartilage formation. These results indicate that proliferating chondrocytes and round osteoblasts produce follistatin, and that the activity of activin is regulated by changes in the expression of follistatin at the stages of chondrogenesis and transition from cartilage to bone.

Decreased follistatin gene expression in gonadotroph adenomas.

What growth factors are involved in the pathogenesis of gonadotroph adenomas is not yet known. Activin is one possible candidate because it stimulates growth and differentiation in many cells, including the gonadotroph cell, and it stimulates FSH secretion, characteristic of gonadotroph adenomas. As activin beta B-subunit is expressed in gonadotroph adenomas, we sought to determine whether activin receptor II and follistatin are also expressed. Total ribonucleic acid (RNA) was extracted from 10 gonadotroph adenomas that did not express pit-1 and was reverse transcribed. The resulting complementary DNAs for human activin receptor II and follistatin were amplified by PCR. All 10 adenomas expressed activin receptor II messenger RNA (mRNA), as did nonadenomatous pituitary tissue. Only 2 of the 10 gonadotroph adenomas expressed detectable follistatin mRNA, although all 4 nonadenomatous pituitaries did. Quantitation of follistatin mRNA by competitive reverse transcription-PCR showed that none of the 10 gonadotroph adenomas expressed as much follistatin mRNA as did the 4 nonadenomatous pituitaries, and 8 of the 10 expressed less than 10% as much. Immunospecific staining showed follistatin in the cytoplasm of the gonadotroph cells of all 5 nonadenomatous pituitaries studied, but only faintly in 1 gonadotroph adenoma and not at all in the other 9. These results suggest that pit-1-negative gonadotroph adenomas express less follistatin mRNA and follistatin peptide than do nonadenomatous gonadotroph cells. A consequence could be less binding, and thereby enhanced effectiveness, of activin, contributing to adenoma growth.

Unique recognition of activin and inhibin by polyclonal antibodies to inhibin subunits.

Inhibin-A is a glycoprotein composed of an alpha subunit containing a glycosylation site and a beta A subunit, whereas activin-A is a homodimer of two inhibin beta A subunits. We examined the recognition of activin-A and inhibin-A by several antisera to the alpha or beta A subunit, and factors affecting the recognition. A total of six polyclonal antibodies to inhibin subunits, i.e., two antisera to a peptide fragment of the alpha subunit [alpha (1-19) and alpha (1-26)], and four antisera to the beta A subunit [beta A (1-10), beta A (70-79), beta A (87-99), and beta A (94-105)], was generated. On Western blot analysis, the anti-beta A (87-99) and beta A (94-105) sera recognized recombinant human activin-A but not inhibin-A under non-reducing conditions. When inhibin-A was deglycosylated with N-glycosidase-F, inhibin-A could be recognized by the anti-beta A (87-99) and beta A (94-105) sera. In addition, when activin-A bound to a nitrocellulose membrane was pre-incubated with recombinant human follistatin, the recognition of activin-A by the anti- beta A (87-99) and beta A (94-105) sera was decreased. These results suggested that the lower affinity of follistatin to inhibin-A than to activin-A might be likely explained as reflecting a site associated with the glycosylation of inhibin-A. However, the exposure of amino acids 87-105 of the inhibin beta A subunit on the molecular surface through deglycosylation did not increase the affinity of inhibin-A for follistatin but rather resulted in poor binding with follistatin. The present data suggest that (1) amino acids 87-105 of the inhibin/activin beta A subunit are located on the molecular surface, although this region of inhibin-A is concealed by the carbohydrate chain of the alpha subunit, (2) the region responsible for follistatin binding within the activin beta A subunit is spanned by amino acids 87-105, and (3) the mode of binding of inhibin-A to follistatin is quite different from that of activin-A to follistatin, and the former may be influenced by glycosylation.

Regulation of Activin Type‐II Receptor mRNA Levels in Rat Hypothalamus by Estradiol in vivo

A solution-hybridization S1-nuclease protection assay was used to evaluate the expression of messenger RNAs for the activin beta A subunit and type II activin receptor in adult rat brain. Results indicate the presence of beta A subunit mRNA in both hypothalamus and brainstem, with approximately two-fold higher levels in brainstem. Levels of activin type II receptor mRNA were similar in the hypothalamus of young virgin and 15-day lactating females, and in females in which pups were removed after a 5-day lactation period. Male rats castrated prepubertally (30 days p.n.) had approximately 220% higher (P < 0.05) hypothalamic activin type II receptor mRNA levels than postpubertal, 3-month old age-matched sham controls. Two month treatment of castrate rats with estradiol (200 ng/g, i.p. every 2 days) reduced hypothalamic activin type II receptor mRNA expression to control levels; the same dose of testosterone had no effect. The expression of the hypothalamic activin type II receptor gene may be estrogen-regulated in vivo.

Tumor-specific expression and alternate splicing of messenger ribonucleic acid encoding activin/transforming growth factor-beta receptors in human pituitary adenomas.

Activin, a member of the transforming growth factor-beta (TGF beta) cytokine family, acts as a pituitary cell mitogen via a novel family of receptor-linked serine/threonine (Ser/Thr) kinases. Pituitary tumors synthesize activin subunits, and the autocrine action of these growth factors may modulate tumor proliferation. We, therefore, investigated the expression of activin/TGF beta type I receptor messenger ribonucleic acids (mRNAs), designated ALK1 through ALK5 (ALK = activin receptor-like kinase), and type II receptor mRNAs using RT-PCR in 34 human pituitary adenomas of all phenotypes and normal pituitary tissue. ALK2 and ALK5, specific mediators of activin and TGF beta signals, respectively, were found to be expressed only in tumor and not in normal pituitary cells, and ALK2 expression was found only in tumors of a mammosomatotroph cell lineage. ALK1, ALK3, and ALK4 mRNAs were found in both normal and neoplastic pituitary cells. The alternatively spliced cytoplasmic domain of ALK4 consists of 11 kinase subdomains, that are critical for modulating receptor function and intracellular signaling. Truncated forms of the ALK4 cytoplasmic domain lacking these subdomains may attenuate activin signal transduction and affect both tumor phenotype and proliferation via the formation of inactive type I/type II complexes. Three truncated ALK4 receptor mRNAs generated by alternate splicing of the cytoplasmic Ser/Thr kinase domain were found to be tumor specific. One of these truncated receptor mRNAs, ALK4-5, is a novel splice variant that has not been previously described. Expression of the ActRII and T beta RII type II receptor mRNAs, which specifically bind activin and TGF beta, respectively, was highly prevalent among all tumor subtypes and normal pituitary tissue. However, ActRIIB, an activin-specific type II receptor that displays a 3- to 4-fold higher affinity for ligand than ActRII, was expressed in 94% of tumors, but was not prevalent in normal tissue. These data are the first to demonstrate tumor-specific expression of Ser/Thr kinase receptors mRNAs and their splice variants in human pituitary adenomas.

Characterization of inhibin/activin subunit, follistatin, and activin type II receptors in human ovarian cancer cell lines: a potential role in autocrine growth regulation.

Although ovarian cancer is the most common gynecological malignancy with a relatively poor 5-yr survival record, the mechanism(s) by which these tumors arise is not well understood. A role for inhibins and activins in regulating this transformation is suggested by the detection of circulating alpha or dimeric inhibin in some patients with ovarian cancer and by the alpha inhibin knockout mouse, in which development of gonadal tumors in 100% of homozygotes is associated with greatly elevated activin levels. To develop diagnostic tools with greater specificity for ovarian cancers, the present study was targeted at characterizing the biosynthetic capacity of the epithelial ovarian cancer cell lines from the American Type Culture Collection with respect to inhibin, activin, the related activin-binding protein follistatin (FS), and activin receptor type II. In addition, the functional capacity of this system was investigated by examining the ability of activin and FS to modulate cellular proliferation. All six cell lines contained abundant messenger RNA (mRNA) for activin receptor type II, but no inhibin alpha-subunit mRNA was detected in any cell line. Two cell lines contained mRNA for activin beta B-subunit (CaOV4 and SKOV3), one cell line contained beta A-subunit mRNA (SW626), and one cell line contained both (ES2); the latter also contained FS mRNA. FS mRNA was detected in another cell line (PA-1) that contained no detectable activin beta-subunit mRNA. Finally, one cell line (CaOV3) contained neither beta-subunit nor FS mRNA. Protein secretion was also examined. Consistent with the mRNA studies, the two cell lines containing FS mRNA secreted FS (PA-1 and ES2 cells), whereas three of the remaining lines secreted activin (A or B). In the cell line containing neither FS nor beta-subunit mRNA, no FS or activin could be detected. Finally, none of the cell lines secreted detectable immunoreactive inhibin. The effects of exogenous activin and FS on cellular proliferation were examined in these cell lines. No response was detected in the two cell lines that secreted FS (PA-1 and ES2). For the four cell lines not synthesizing FS, treatment with activin (1-100 ng/ml) resulted in an increase, whereas FS treatment (1-100 ng/ml) resulted in a decrease in cellular proliferation, as determined by [3H]thymidine incorporation. The response to activin correlated negatively with endogenous activin production, suggesting that autocrine activin production may be involved with cell proliferation. The differential expression and production of inhibin/activin subunits, activin receptors, and follistatin as well as the range of responses to exogenous activin among six ovarian epithelial cancer cell lines suggest that this family of hormones may be important in regulating cell proliferation in the ovary. Whether primary tumors have the same profile and the degree to which these results can be generalized to additional forms of ovarian cancer remain to be determined.

Induction of β-A activin expression by synaptic activity and during neocortical development

beta-A activin is a member of the transforming growth factor-beta family and has been implicated in nerve cell survival and inhibition of differentiation in vitro [Hashimoto M. et al. (1990) Biochem. biophys. Res. Commun. 173, 193-200; Schubert D. et al. (1990) Nature 344, 868-870]. In our studies to identify genomic mechanisms involved in long-term neuronal responses to synaptic activity, we have determined that beta-A activin messenger RNA is rapidly and transiently induced in neurons of the adult rat brain by excitatory synaptic input. Synaptic mechanisms involved in beta-A activin messenger RNA induction were examined in adult hippocampus and cortex using the long-term potentiation paradigm. beta-A activin messenger RNA is induced in granule cell neurons of the hippocampus by high-frequency synaptic stimuli that produce long-term potentiation, and this induction is blocked by the N-methyl-D-aspartate type glutamate receptor antagonist, dizocilpine. beta-A activin messenger RNA is expressed at basal levels in neurons of layers II/III and V/VI, and this expression rapidly decreases following sensory deafferentation of the visual cortex or systemic administration of dizocilpine, suggesting that beta-A activin expression is regulated by physiological excitatory synaptic activity. In developing brain, beta-A activin is expressed in the neocortex and neostriatum beginning at embryonic day 17. beta-A activin expression in late fetal cortex is enriched in postmitotic neurons at the lower boundary of the dense cortical plate. As development progresses, beta-A activin expression continues to be enriched in neurons at the boundary between the hypercellular cortical plate and the subjacent, more mature deep layers. This inside-out progression of beta-A activin expression follows the well-characterized radial gradient of cortical development. Expression of beta-A activin messenger RNA is rapidly regulated in early postnatal cortex and striatum by GABA and glutamate antagonists, suggesting that beta-A activin is also regulated as a rapid response gene in developing brain, and that the high basal levels reflect a steady-state response to developmental signals. Since activin receptors are enriched in neurons of developing and adult brain [Cameron V. A. et al. (1994) Endocrinology 134, 799-808; Roberts V. J. and Barth S. L. (1994) Endocrinology 134, 914-922], our observations suggest a role for activin signaling in neuronal responses to synaptic and developmental activity. In this study, we analyse the induction of expression of beta-A activin, a member of the transforming growth factor-beta family of secreted peptides, in response to synaptic activity and in the developing brain. The elevated and specific expression of beta-A activin during fetal and early postnatal neocortical development and its later regulation by excitatory activity postnatally and in the adult suggests that the activin signaling pathway functions at multiple developmental stages in the neuroplastic response.

EXPRESSION OF ACTIVIN AND ACTIVIN RECEPTORS IN HUMAN PROSTATIC-CARCINOMA CELL-LINE DU145

The purpose of this study was to determine whether DU145, a human prostate cancer cell line: (a) transcribes mRNAs coding for beta A- and beta B-subunits of activin, a member of transforming growth factor beta (TGF beta) superfamily, and activin receptors I, II, and IIB; and (b) produces activin proteins. The expression and localization of the mRNAs were elucidated by the reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization techniques. The production of activin was determined by immunocytochemistry. We have observed that messenger RNAs encoding activin beta A-, beta B-subunits, and activin receptors I, II, and IIB, but not that of the alpha-subunit of inhibin, were expressed, and activin proteins, but not inhibin, were produced, by DU145 cells. Furthermore, the RT-PCR products were confirmed by DNA sequencing. It is concluded that activins and their receptors are expressed in DU145 and activins may have autocrine functions in DU145 cells.

Development and characterization of a simian virus 40-transformed, temperature-sensitive rat antimesometrial decidual cell line

The rat decidual tissue is formed by two cell populations, which express different genes and play diverse roles in pregnancy. Cells that decidualize in the antimesometrial region secrete several hormones and serve as a true endocrine gland. Isolation and maintenance of these decidual cells in primary culture is difficult. The goal of these experiments was to develop a cell line to serve as a model to study the expression and regulation of various genes specific to the antimesometrial decidual cells. Decidualization was induced in pseudopregnant rats. The antimesometrial decidua was dissected out, and cells were enzymatically dissociated and were cultured for 18 h at 37 C in RPMI-1640 medium containing 10% fetal bovine serum. Cells were washed repeatedly and then infected with a temperature-sensitive mutant of the simian virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified and harvested. Whereas primary cells in culture did not divide, the cloned decidual cell lines demonstrated transformed features; they multiplied at 33 C and formed multilayers. At the nonpermissive temperature (39 C), cell replication decreased, and after 4 days of culture the cells lost their transformed phenotype and continued to grow as a monolayer similar to primary cells. Cells under these conditions also assumed morphological characteristics similar to antimesometrial cells: polynucleated, large, and having cytoplasm filled with lipid droplets. Interestingly, cells cultured at 39 C that were shifted back to 33 C resumed rapid growth. To determine whether these cells also express messenger RNAs (mRNAs) found in normal antimesometrial decidual cells, the presence of activin beta A mRNA was investigated by Northern analysis and reverse transcription polymerase chain reaction. A single 6.8-kilobase activin beta A transcript was expressed abundantly at both 33 and 39 C, indicating that even when cells are rapidly dividing they express activin beta A. Activin beta B mRNA was also expressed in these cells, although in lower abundance, as were two binding proteins for activin, activin receptor II and follistatin. The activin beta A and beta B genes were responsive to cAMP stimulation in these cells. Since the hallmark of the antimesometrial decidual cells is the secretion of PRL-related hormones, the expression of decidual PRL-related protein and PRL-like protein B was examined. Northern analysis revealed a major 1.2-kilobase transcript of PRL-like protein B expressed equally at both temperatures.

Development and characterization of a simian virus 40-transformed, temperature-sensitive rat antimesometrial decidual cell line.

The rat decidual tissue is formed by two cell populations, which express different genes and play diverse roles in pregnancy. Cells that decidualize in the antimesometrial region secrete several hormones and serve as a true endocrine gland. Isolation and maintenance of these decidual cells in primary culture is difficult. The goal of these experiments was to develop a cell line to serve as a model to study the expression and regulation of various genes specific to the antimesometrial decidual cells. Decidualization was induced in pseudopregnant rats. The antimesometrial decidua was dissected out, and cells were enzymatically dissociated and were cultured for 18 h at 37 C in RPMI-1640 medium containing 10% fetal bovine serum. Cells were washed repeatedly and then infected with a temperature-sensitive mutant of the simian virus. Transformed cells were maintained at the permissive temperature (33 C) until colonies were identified and harvested. Whereas primary cells in culture did not divide, the cloned decidual cell lines demonstrated transformed features; they multiplied at 33 C and formed multilayers. At the nonpermissive temperature (39 C), cell replication decreased, and after 4 days of culture the cells lost their transformed phenotype and continued to grow as a monolayer similar to primary cells. Cells under these conditions also assumed morphological characteristics similar to antimesometrial cells: polynucleated, large, and having cytoplasm filled with lipid droplets. Interestingly, cells cultured at 39 C that were shifted back to 33 C resumed rapid growth. To determine whether these cells also express messenger RNAs (mRNAs) found in normal antimesometrial decidual cells, the presence of activin beta A mRNA was investigated by Northern analysis and reverse transcription polymerase chain reaction. A single 6.8-kilobase activin beta A transcript was expressed abundantly at both 33 and 39 C, indicating that even when cells are rapidly dividing they express activin beta A. Activin beta B mRNA was also expressed in these cells, although in lower abundance, as were two binding proteins for activin, activin receptor II and follistatin. The activin beta A and beta B genes were responsive to cAMP stimulation in these cells. Since the hallmark of the antimesometrial decidual cells is the secretion of PRL-related hormones, the expression of decidual PRL-related protein and PRL-like protein B was examined. Northern analysis revealed a major 1.2-kilobase transcript of PRL-like protein B expressed equally at both temperatures.(ABSTRACT TRUNCATED AT 250 WORDS)

Monocyte-induced cytokine expression in cultured human retinal pigment epithelial cells

Monocytes and retinal pigment epithelial cells are intimately associated in membranes of eyes with proliferative vitreoretinopathy and in certain types of uveitis. The goal of this study was to determine whether monocytes modulate cytokine expression in retinal pigment epithelial cells, and if so, to identify the monocyte products responsible for this effect. Cultured human retinal pigment epithelial cells were exposed to varying concentrations of monocyte-conditioned medium from unstimulated human monocytes for 1-48 hr, or from monocytes prestimulated with lipopolysaccharide. mRNA expression of interleukin-1 beta, interleukin-6, interleukin-8, melanoma growth stimulating activity/gro alpha and gamma, macrophage colony stimulating factor, transforming growth factor-beta 2, basic fibroblast growth factor and activin beta A chain was determined by reverse transcription polymerase chain reaction. Protein secretion of selected cytokines, interleukin-1 beta, interleukin-6, interleukin-8, macrophage colony stimulating factor and transforming growth factor-beta 2 was measured in RPE-conditioned medium by ELISA. Retinal pigment epithelial cells constitutively expressed mRNA for interleukin-6, macrophage colony stimulating factor, transforming growth factor-beta 2, basic fibroblast growth factor and activin beta A chain. Interleukin-1 beta, melanoma growth stimulating activity/gro alpha and gamma and interleukin-8 were not expressed under basal conditions. Stimulated monocyte-conditioned medium markedly induced mRNA of all cytokines except basic fibroblast growth factor and transforming growth factor-beta 2 in a dose- and time-dependent manner. Unstimulated monocyte-conditioned medium was a less potent inducing agent, but still enhanced mRNA expression of interleukin-6, interleukin-8 and melanoma growth stimulating activity/gro alpha. Stimulated monocyte-conditioned medium also induced a time-dependent increase in interleukin-6, Interleukin-8, macrophage colony stimulation factor and transforming growth factor-beta 2, but not interleukin-1 beta protein secretion (p < 0.05 for all time points). Neutralizing antibodies to interleukin-1 beta, or tumour necrosis factor alpha, but not interleukin-1 alpha, significantly reduced cytokine mRNA expression induced by stimulated monocyte-conditioned medium. The combination of all three neutralizing antibodies almost entirely eliminated monocyte-induced mRNA expression and protein production of all cytokines studied. Activated monocytes secrete a heterogeneous mixture of products that together strongly induce expression of multiple cytokines in human retinal pigment epithelial cells. Most if not all of the inducing effect can be accounted for by interleukin-1 beta and tumour necrosis factor alpha. Because cytokines have been implicated in proliferative vitreoretinopathy and uveitis, monocyte-mediated cytokine expression by RPE cells may serve to initiate and perpetuate these diseases.

Activin beta A- and beta B-subunit expression in the developing chicken bursa of fabricius.

The bursa of Fabricius (BF) is a site for B-lymphocyte maturation in birds. The BF is also known to function as an endocrine organ, with regulatory effects on steroid-secreting glands (testes, ovaries, and adrenal glands) during embryonic development. To study the possible involvement of the growth and differentiation factors, inhibins and activins, in bursal differentiation and function, the expression of inhibin/activin subunits in chicken BF was examined using specific antibodies against inhibin/activin alpha-, beta A-, and beta B-subunits. Bursae from chickens from 11 days of embryonic development until 22 weeks after hatching were studied. Immunoreactive beta A- and beta B-subunits were demonstrated in bursal epithelial cells throughout the time period studied. Immunoreactivities for both subunits were most intense and widespread from day 18 of embryonation until 1 week after hatching. Inhibin alpha-subunits were not detected. Partially different locations were shown for beta A- and beta B-subunits, suggesting different roles for activin-A (beta A beta A homodimer) and activin-B (beta B beta B homodimer) in bursal development and function. As activin beta A-subunit immunoreactivity was predominantly localized in medullary epithelia, it may be assumed that the developing B-cells within follicular medullae might be the target for activin-A action in the chicken BF. The most conspicuous site for activin-B production, on the other hand, was the follicle-associated epithelium, which is able to take up antigens from the cloacal environment and pass them to medullary cells. The data suggest that activin-A and -B may have different roles in modulating bursal microenvironment during B-lymphocyte differentiation. The possible role of bursal activins in the regulation of steroidogenesis in birds is discussed.