Amy L. Blount

Betaglycan binds inhibin and can mediate functional antagonism of activin signalling

Activins and inhibins, structurally related members of the TGF-β superfamily of growth and differentiation factors, are mutually antagonistic regulators of reproductive and other functions. Activins bind specific type II receptor serine kinases (ActRII or IIB) to promote the recruitment and phosphorylation of the type I receptor serine kinase, ALK4 (refs 7,8,9), which then regulates gene expression by activating Smad proteins. Inhibins also bind type II activin receptors but do not recruit ALK4, providing a competitive model for the antagonism of activin by inhibin. Inhibins fail to antagonize activin in some tissues and cells, however, suggesting that additional components are required for inhibin action. Here we show that the type III TGF-β receptor, betaglycan, can function as an inhibin co-receptor with ActRII. Betaglycan binds inhibin with high affinity and enhances binding in cells co-expressing ActRII and betaglycan. Inhibin also forms crosslinked complexes with both recombinant and endogenously expressed betaglycan and ActRII. Finally, betaglycan confers inhibin sensitivity to cell lines that otherwise respond poorly to this hormone. The ability of betaglycan to facilitate inhibin antagonism of activin provides a variation on the emerging roles of proteoglycans as co-receptors modulating ligand–receptor sensitivity, selectivity and function.

Autocrine/paracrine regulation of pituitary function by activin, inhibin and follistatin.

The precise regulation of the anterior pituitary is achieved by the cell-specific and combined actions of central, peripheral and local factors. Activins, inhibins, and follistatins were first discovered as gonadal factors with actions on FSH production from pituitary gonadotropes. With the realization that these factors are expressed in a wide array of tissues, including the pituitary, it became apparent that the functional importance of activins, inhibins, and follistatins extends beyond the reproductive axis and that they often exert their effects by autocrine/paracrine mechanisms. As members of the TGF-beta superfamily, activins and inhibins control and orchestrate many physiological processes and are vital for the development, the growth, and the functional integrity of most tissues, including the pituitary. Activins exert effects on multiple pituitary cell types but the best-characterized pituitary targets of the autocrine/paracrine function of activins are the gonadotropes. The autocrine/paracrine function of the activin-binding proteins, follistatins, constitutes an important local mechanism to modulate activin bioactivity while the restricted actions of gonadal inhibins to betaglycan-expressing gonadotropes provides a secondary mode of regulation of cell-specific actions of activins. The aim of this review is to highlight and evaluate experimental evidence that supports the roles of activins, inhibins, and follistatins as autocrine, paracrine, and/or endocrine modulators of the pituitary.

Urocortin Messenger Ribonucleic Acid: Tissue Distribution in the Rat and Regulation in Thymus by Lipopolysaccharide and Glucocorticoids*

Urocortin (Ucn), a new mammalian member of the CRF family, is a candidate endogenous ligand for type 2 CRF receptors. In a survey of peripheral tissues from adult male rats, we found that Ucn messenger RNA (mRNA) was abundant in the gastrointestinal tract and immune tissues such as thymus and spleen. We next tested the hypothesis that levels of Ucn mRNA levels in thymus and spleen would be altered after immune activation. As measured by ribonculease protection assay, lipopolysaccharide (LPS) induced a 2-fold time-dependent increase in thymic Ucn mRNA levels within 6 h. By contrast, splenic Ucn mRNA levels decreased after LPS. Because LPS activates the hypothalamus-pituitary-adrenal (HPA) axis, we examined whether the effects of LPS on Ucn mRNA might be mediated through changes in HPA axis hormones. Ucn mRNA in thymus, but not spleen, was significantly increased after ACTH injection; however, LPS did not increase Ucn expression in the thymus of adrenalectomized rats with corticosterone replacement, despite substantial increases in ACTH. Finally, sc injection of corticosterone stimulated Ucn mRNA comparably to that of LPS. Together, these results suggest that Ucn mRNA expression can increase after immune activation in a corticosterone-dependent manner, and that such changes in Ucn mRNA may be an additional consequence of HPA axis activation.

FoxL2 and Smad3 coordinately regulate follistatin gene transcription

Follistatin is a transcriptional target and a modulator of activin action. Through an autocrine/paracrine loop, activin controls follistatin levels and thus regulates its own bioavailability. In gonadotropic αT3-1 cells, activin induces follistatin transcription primarily through the action of Smad3 at an intronic Smad-binding element (SBE1). Using a proteomics approach, we searched for endogenous αT3-1 proteins that participate in SBE1-mediated transcription. We identified FoxL2, a member of the forkhead family, as a candidate modulator of SBE1 function. Mutations of FoxL2 are associated with the blepharophimosis/ptosis/epicanthus inversus syndrome characterized with craniofacial defects and premature ovarian failure. FoxL2 localizes to α-glycoprotein subunit- and follicle-stimulating hormone β-positive cells of the adult mouse pituitary and is present in αT3-1 and LβT2 cells, but its pituitary actions remain largely unknown. We have determined that FoxL2 binds to a forkhead-binding element (FKHB) located just downstream of the SBE1 site of the follistatin gene and functions as a Smad3 partner to drive SBE1-mediated transcription in αT3-1 cells treated with activin. Chromatin immunoprecipitation assays confirm that endogenous FoxL2 and Smad3 are recruited to the intronic enhancer of the follistatin gene where the SBE1 and FKHB sites are located. Exogenous FoxL2 enhances SBE1-mediated transcription, and short hairpin RNA-mediated knockdown of endogenous FoxL2 protein compromises this effect in αT3-1 cells. FoxL2 directly associates with Smad3 but not Smad2 or Smad4. This association between Smad3 and FoxL2 is mediated by the MH2 domain of Smad3 and is dependent on an intact forkhead domain in FoxL2. Altogether, these observations highlight a novel role for FoxL2 and suggest that it may function as a transcriptional regulator and a coordinator of Smad3 targets.

Regulation and Actions of Smad7 in the Modulation of Activin, Inhibin, and Transforming Growth Factor-β Signaling in Anterior Pituitary Cells1

Activins and transforming growth factor-β (TGFβ) are crucial autocrine, paracrine, and endocrine modulators of anterior pituitary function. Activins regulate most pituitary cells and lactotropes are targets of TGFβ. Smad2 and Smad3 are two cellular mediators of activin/TGFβ signaling, whereas Smad7 is as an inducible, negative modulator of the pathway. This study was undertaken to evaluate Smad7 regulation in the pituitary. Activin A rapidly and transiently increased Smad7 messenger RNA (mRNA) levels of rat anterior pituitary (RAP), clonal gonadotrope (αT3–1 and LβT2), and corticotrope (AtT20) cells with an EC50 of 0.1–0.2 nm. In RAP cells, activin A or TGFβ1 had equivalent effects that were additive. Follistatin, known to bind and inactivate activins, prevented Smad7 induction by activin. Inhibin A partially antagonized activin A, perhaps reflecting gonadotrope-selective actions. This antagonism was also evident with αT3–1 and LβT2 gonadotropes. Forskolin had no measurable effect in RAP cells, but increa...

B cell antigen receptors of the IgM and IgD classes are clustered in different protein islands that are altered during B cell activation.

Antigen stimulates the dispersion and remodeling of preformed distinct clusters of B cell receptors on the cell surface. How BCRs mingle The B cell antigen receptor (BCR) consists of a plasma membrane–bound antibody [immunoglobulin (Ig)] that is associated with a pair of signaling proteins. Antigen binding to the BCR stimulates B cells to differentiate into antibody-secreting cells. Maity et al. used high-resolution microscopy, electron microscopy, and proximity ligation assays to visualize the organization of IgM-BCRs and IgD-BCRs on mature B cells. Under resting conditions, the different BCRs were separated in relatively large clusters called protein islands. Antigen triggered the protein islands to become smaller and more disperse, reducing the distance between the different BCRs. The B cell antigen receptors (BCRs) play an important role in the clonal selection of B cells and their differentiation into antibody-secreting plasma cells. Mature B cells have both immunoglobulin M (IgM) and IgD types of BCRs, which have identical antigen-binding sites and are both associated with the signaling subunits Igα and Igβ, but differ in their membrane-bound heavy chain isoforms. By two-color direct stochastic optical reconstruction microscopy (dSTORM), we showed that IgM-BCRs and IgD-BCRs reside in the plasma membrane in different protein islands with average sizes of 150 and 240 nm, respectively. Upon B cell activation, the BCR protein islands became smaller and more dispersed such that the IgM-BCRs and IgD-BCRs were found in close proximity to each other. Moreover, specific stimulation of one class of BCR had minimal effects on the organization of the other. These conclusions were supported by the findings from two-marker transmission electron microscopy and proximity ligation assays. Together, these data provide evidence for a preformed multimeric organization of BCRs on the plasma membrane that is remodeled after B cell activation.

Actions of activins, inhibins and follistatins: implications in anterior pituitary function.

1. The anterior pituitary is well documented to be under the control of central and peripheral factors that dynamically interact to affect cell‐specific modulation of pituitary functions. However, it is becoming increasingly evident that these extrinsic factors work in concert with a variety of local products that exert autocrine/paracrine control on pituitary cells.

Regulation of follicle-stimulating hormone secretion by the interactions of activin-A, dexamethasone and testosterone in anterior pituitary cell cultures of male rats

This study was designed to evaluate the effects of glucocorticoids and gonadal steroids on the expression of inhibin/activin subunits and follistatin of the anterior pituitary and test the hypothesis that resulting changes in the local activin/inhibin/follistatin tone contribute to steroid effects on follicle stimulating hormone (FSH) production from gonadotropes. In primary cell cultures of male rat anterior pituitaries, dexamethasone (DEX) or testosterone (T) stimulated FSH secretion and FSHβ mRNA and their effects were additive with activin-A. Follistatin (FS288) and inhibin-A antagonized the rise in FSH secretion both in the absence and presence of exogenous activin-A. Despite the similarity in their action on FSH production, DEX and T had opposite effects on follistatin mRNA levels. Follistatin mRNA levels of cultured rat anterior pituitary cells were elevated upon the addition of DEX but attenuated by T. On the other hand, both DEX and T suppressed inhibin/activin βB mRNA levels while only DEX affected βA mRNA. In these cells, activin-A stimulated follistatin and inhibin/activin βB mRNA levels but had no effect on βA. Together, DEX and activin-A caused a further increase in follistatin mRNA levels while T attenuated the effect of activin-A alone. Both steroids attenuated the effect of activin-A on βB mRNA accumulation. These results support the possibility that DEX and T, possibly acting on different subsets of anterior pituitary cells, use distinct mechanisms to modify the local activin/inhibin/follistatin circuitry and thereby upregulate FSH production from the anterior pituitary gonadotropes.

A Smad-binding Element in Intron 1 Participates in Activin-dependent Regulation of the Follistatin Gene

Follistatins exert critical autocrine or paracrine control in many tissues by binding and bio-neutralizing activin and several other transforming growth factor-β ligands. In the pituitary, activin acts locally to induce follistatin expression and thus modulate its own actions. This local feedback loop safeguards against excessive activin signaling and maintains the necessary balance of activin and follistatin tone. To better understand the mechanisms underlying the activation of follistatin by activin A, follistatin transcription was evaluated in gonadotrope-derived αT3-1 cells. Transient transfection experiments established that follistatin-luciferase plasmids that incorporate up to 2.86 kb of the upstream region of the rat follistatin gene are not induced by activin A in αT3-1 cells. On the other hand, plasmids that incorporate intron 1 are responsive to activin A and induced by a constitutively active form of ALK4. These experiments ultimately identified a conserved Smad-binding element (SBE1) in intron 1, between +1791 and +1795. In αT3-1 cells treated with activin A, SBE1 preferentially recruits Smad3, but not Smad2, and mediates Smad3-dependent activation of follistatin transcription. shRNA knockdown of endogenous Smad3 in these cells compromises SBE1-mediated transcription in response to activin A and interferes with its ability to positively regulate follistatin mRNA levels. The findings of the current work illustrate the critical role of intron 1 of the follistatin gene in mediating Smad-dependent effects of activin and regulating the expression level of this gene in some cell types, such as pituitary cells of gonadotrope lineage.

Impaired FSHβ Expression in the Pituitaries of Foxl2 Mutant Animals

Forkhead box L2 (FoxL2) is required for ovarian development and differentiation. FoxL2 is also expressed in the pituitary where it has been implicated in the development and regulation of gonadotropes, which secrete LH and FSH, the endocrine signals that regulate folliculogenesis in the ovary and spermatogenesis in the testis. Here, we show that FoxL2 is not required for the specification of gonadotropes; the pituitaries of Foxl2 mutant mice contain normal numbers of gonadotropes that express glycoprotein α subunit and LHβ. Whereas the specification of gonadotropes and all other hormonal cell types is normal in the pituitaries of Foxl2 mutant animals, FSHβ levels are severely impaired in both male and female animals, suggesting that FoxL2 is required for normal Fshb expression. The size of the pituitary is reduced in proportion to the smaller body size of Foxl2 mutants, with a concomitant increase in the pituitary cellular density. In primary pituitary cultures, activin induces FSH secretion and Fshb mRNA expression in cells from wild-type mice. In cells from Foxl2 mutant mice, however, FSH secretion is not detected, and activin is unable to drive Fshb expression, suggesting that the mechanism of activin-dependent activation of Fshb transcription is impaired. However, a small number of gonadotropes in the ventromedial region of the pituitaries from Foxl2 mutant mice maintain FSHβ expression, suggesting that a FoxL2- and activin-independent mechanism can drive Fshb transcription. These data indicate that, in addition to its role in the ovary, FoxL2 function in the pituitary is required for normal expression of FSH.