M Erämaa

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.

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.

Transforming growth factor-β1 and -β2 induce inhibin and activin βB-subunit messenger ribonucleic acid levels in cultured human granulosa-luteal cells *

Objective To examine the effect of transforming growth factor- β (TGF- β ) 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- β 1 or TGF- β 2 and/or hCG whereafter cellular RNA was extracted for Northern or dot blot filter hybridization with inhibin α -, β A , and β B -subunit complementary DNA probes. Results Both TGF- β 1 and TGF- β 2 induced the expression of a 4.8-kb inhibin and activin β B -subunit messenger (mRNA) transcript in a time- and dose-dependent manner but had no effect on α - β A -subunit mRNA levels. Human chorionic gonadotropin alone did not affect β B -subunit mRNA levels, but when administered together with TGF- β s, it prevented the induction of β B -subunit mRNAs. Conclusions Our results suggest that in human ovary, granulosa, or thecal cell-derived TGF- β 1 or - β 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 β B -subunit. The effect of TGF- β is clearly different from that of gonadotropins, which potently induce the α - and β A -subunit mRNAs, indicating that distinct components of the human ovarian inhibin and activin system are regulated differentially by endocrine and local factors.

Differential regulation of inhibin/activin α- and βA-subunit and follistatin mRNAs by cyclic AMP and phorbol ester in cultured human granulosa-luteal cells

Abstract Granulosa cell-derived inhibin A (a dimer of α - and β A -subunits), activin A (a homodimer of β 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 α - and β A -subunit and follistatin mRNAs in cultured human granulosa-luteal cells. 8br-cAMP induced α - and β A -subunit and follistatin steady-state mRNA levels in a time- and concentration-dependent manner. The levels of α-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, β 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 α-subunit mRNA levels but it rapidly induced β 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 β A -subunit and follistatin mRNA levels synergistically at 3 h. By contrast, TPA had a potent inhibitory effect on 8br-cAMP- and hCG-induced α-subunit levels. Neither 8br-cAMP nor TPA regulated inhibin/activin β 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 α- and β 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.

Co-ordinate expression of activin A and its type I receptor mRNAs during phorbol ester-induced differentiation of human K562 erythroleukemia cells

Activins were originally isolated based on their ability to stimulate follicle-stimulating hormone secretion but later they have been shown to regulate a number of different cellular functions such as nerve cell survival, mesoderm induction during early embryogenesis as well as hematopoiesis. We studied the regulation of activin A, a homodimer of betaA-subunits, mRNA and protein in K562 erythroleukemia cells, which are known to be induced toward the erythroid lineage in response to activin or TGF-beta or toward the megakaryocytic lineage by the phorbol ester protein kinase C activator 12-O-tetradecanoylphorbol-13-acetate (TPA). Here we show by Northern blot analysis as well as by Western and ligand blotting that TPA strongly promotes activin betaA-subunit mRNA and activin A protein expression in K562 cells in time- and concentration dependent manner. In contrast, neither activin A nor TGF-beta induced betaA-subunit mRNA expression during erythroid differentiation in K562 cells. Interestingly, whereas activin type II receptors are not regulated during K562 cell differentiation (Hilden et al. (1994) Blood 83, 2163-2170), we now show that the activin type I and IB receptor mRNAs are clearly induced by TPA but not by activin or TGF-beta. We also show that the inducing effect of TPA on expression of activin betaA-subunit mRNA is potentiated by the protein kinase A activator 8-bromo-cAMP. We conclude that activin A and its type I receptors appear to be co-ordinately up-regulated during megakaryocytic differentiation of K562 cells.