A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli.

Cell-surface-receptor pathways amplify weak, rare and local stimuli to induce cellular responses. This task is accomplished despite signaling components that segregate into nanometer-scale membrane domains. Here we describe a 'catch-and-release' mechanism that amplified and dispersed stimuli by releasing activated kinases from receptors lacking intrinsic catalytic activity. Specifically, we discovered a cycle of recruitment, activation and release for Zap70 kinases at phosphorylated T cell antigen receptors (TCRs). This turned the TCR into a 'catalytic unit' that amplified antigenic stimuli. Zap70 released from the TCR remained at the membrane, translocated, and phosphorylated spatially distinct substrates. The mechanisms described here are based on widely used protein domains and post-translational modifications; therefore, many membrane-associated pathways might employ similar mechanisms for signal amplification and dispersion.

T cell receptor dwell times control the kinase activity of Zap70.

Kinase recruitment to membrane receptors is essential for signal transduction. However, the underlying regulatory mechanisms are poorly understood. We investigated how conformational changes control T cell receptor (TCR) association and activity of the kinase Zap70. Structural analysis showed that TCR binding or phosphorylation of Zap70 triggers a transition from a closed, autoinhibited conformation to an open conformation. Using Zap70 mutants with defined conformations, we found that TCR dwell times controlled Zap70 activity. The closed conformation minimized TCR dwell times and thereby prevented activation by membrane-associated kinases. Parallel recruitment of coreceptor-associated Lck kinase to the TCR ensured Zap70 phosphorylation and stabilized Zap70 TCR binding. Our study suggests that the dynamics of cytosolic enzyme recruitment to the plasma membrane regulate the activity and function of receptors lacking intrinsic catalytic activity.

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

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 Igß, 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.

Urocortin II gene is highly expressed in mouse skin and skeletal muscle tissues: localization, basal expression in corticotropin-releasing factor receptor (CRFR) 1- and CRFR2-null mice, and regulation by glucocorticoids.

Peptides encoded by the Urocortin (Ucn) II gene, also known as stresscopin-related peptide, were recently identified as new members of the corticotropin-releasing factor (CRF) family. Ucn II is a specific ligand for the type 2 CRF receptor (CRFR). We have demonstrated the peripheral distribution of mouse Ucn (mUcn) II transcripts by using specific mUcn II ribonuclease protection assays, RT-PCR, Southern hybridization, and DNA sequencing. Although Ucn II mRNA is widely expressed in a variety of peripheral tissues, we found it to be most highly expressed in the skin and skeletal muscle tissues. Using a specific RIA for mUcn II, we detected Ucn II-like immunoreactivity (ir) in acid extracts of mouse brain, muscle, and skin. Immunohistochemical studies revealed Ucn II-like ir in both skin epidermis and adnexal structures and in the skeletal muscle myocytes. Ucn II mRNA and ir were also observed in neonatal skeletal muscle cultures in which Ucn II was localized to the myotube. We found a significant increase in Ucn II mRNA levels in the skin, but not in skeletal muscle, of both CRFR1- and CRFR2-null mice compared with their wild-type littermates. We showed that administration of dexamethasone to mice resulted in a decrease of Ucn II mRNA levels in the back skin region 12 h after ip injections. Removal of the adrenal gland significantly increased the levels of Ucn II mRNA in the skin, and the levels were reduced back to normal levels after corticosterone replacement. Further examination of the distribution and regulation of CRFR2 and its specific ligand Ucn II in the skin and skeletal muscle tissues may reveal the manner by which the CRFR2 pathway is involved in the physiological responses to stress in these tissues and in other pathophysiologies of the skin and muscle.

Urocortin III is expressed in pancreatic beta-cells and stimulates insulin and glucagon secretion.

Urocortin (Ucn) III, or stresscopin, is a high affinity ligand for the type 2 corticotropin-releasing factor (CRFR2) receptor recently identified in rodents and human. Ucn III was initially identified as a neuropeptide expressed in discrete areas in the brain. In the present study, we demonstrate that Ucn III is expressed in pancreatic beta-cells and in a mouse beta-cell line, MIN6. Ucn III secretion from the cells was measured using a highly specific RIA, and we found that high potassium, forskolin, or high glucose can stimulate Ucn III secretion from these cells. In vivo studies showed that rats receiving an iv Ucn III injection had a significant elevation of plasma glucagon followed by plasma glucose levels compared with rats receiving vehicle. Ucn III injections also result in an increase in plasma insulin levels. The observed effects of Ucn III were blocked by pretreatment with a CRFR2 antagonist, astressin(2)-B. Furthermore, Ucn III stimulated glucagon and insulin release from isolated rat islets, and astressin(2)-B abolished the effects of Ucn III, in keeping with a CRFR2-mediated mechanism. Taken together, the present studies suggest pancreatic Ucn III acting through CRFR2 is involved in the local regulation of glucagon and insulin secretion.

Rat anterior pituitary folliculostellate cells are targets of interleukin-1beta and a major source of intrapituitary follistatin.

Folliculostellate cells of the anterior pituitary are postulated to be an important source of factors, such as follistatin, that regulate pituitary function by intercellular communication. To gain further insight into the function of this cell type, folliculostellate cells were enriched from cultured rat anterior pituitary cells, and an immortalized cell line designated FS/D1h was established and characterized. These FS/D1h cells express S100 immunoreactivity and produce IL-6 but not pituitary hormones such as GH, ACTH, FSH, and LH. Importantly, FS/D1h cells express large amounts of follistatin mRNA and secrete the protein, as quantified indirectly by the amount of [(125)I]activin A immunoprecipitated with a follistatin antiserum. The FS/D1h cells also express alpha, betaA, and betaB inhibin/activin subunit mRNAs, but whether they produce the corresponding activins and inhibins has not been determined. The response of FS/D1h cells to agents thought to modulate folliculostellate cell function was evaluated. IL-1beta (0.005-5 nM) stimulated the secretion of follistatin and increased mRNA expression. In parallel, IL-6 secretion was stimulated. Dexamethasone, pituitary adenylate cyclase-activating polypeptide(1-27), and lipopolysaccharide but not testosterone, 12-O-tetradecanoylphorbol-13-acetate, or forskolin also increased follistatin secretion. Surprisingly, activin had no effect on follistatin mRNA levels, despite the fact that FS/D1h cells express ActRII, ActRIIB, and ALK-4 (ActRIB). Activin, on the other hand, induced Smad7 mRNA accumulation and exerted an antiproliferative effect on FS/D1h cells. Altogether, these observations support the possibility that follistatin originating from folliculostellate cells participates in mediating the effects of IL-1beta, glucocorticoids, and other agents on the response of pituitary cells to activins.

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.

Impaired FSHbeta 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 LHß. Whereas the specification of gonadotropes and all other hormonal cell types is normal in the pituitaries of Foxl2 mutant animals, FSHß 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 FSHß 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.

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 alphaT3-1 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 alphaT3-1 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 alpha-glycoprotein subunit- and follicle-stimulating hormone beta-positive cells of the adult mouse pituitary and is present in alphaT3-1 and LbetaT2 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 alphaT3-1 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 alphaT3-1 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.

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-beta 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 alphaT3-1 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 alphaT3-1 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 alphaT3-1 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.

Pituitary actions of ligands of the TGF-beta family: activins and inhibins.

Activins, as members of the transforming growth factor-beta superfamily, control and orchestrate many physiological processes and are vital for the development, growth and functional integrity of most tissues, including the pituitary. Activins produced by pituitary cells work in conjunction with central, peripheral, and other local factors to influence the function of gonadotropes and maintain a normal reproductive axis. Follistatin, also produced by the pituitary, acts as a local buffer to bind activin and modulate its bioactivity. On the other hand, inhibins of gonadal origin provide an endocrine feedback signal to antagonize activin signaling in cells that express the inhibin co-receptor, betaglycan, such as gonadotropes. This review highlights the pituitary roles of activin and the mechanisms through which these actions are modulated by inhibin and follistatin.

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 FSHbeta 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 betaB mRNA levels while only DEX affected betaA mRNA. In these cells, activin-A stimulated follistatin and inhibin/activin betaB mRNA levels but had no effect on betaA. 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 betaB 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.