A transcriptionally active pRb-E2F1-P/CAF signaling pathway is central to TGFß-mediated apoptosis.

Transforming growth factor-ß (TGFß) modulates the expression of multiple apoptotic target genes; however, a common and central signaling pathway, acting downstream of TGFß and leading to cell death, has yet to be uncovered. Here, we show that TGFß-induced apoptosis in cancer cells requires the transcription factor E2F1 (E2 promoter-binding factor 1). Using the E2F1 knockout mouse model, we also found E2F1 to be required for TGFß-mediated apoptosis in normal cells. Moreover, we found TGFß to increase E2F1 protein stability, acting at the post-translational level. We further investigated the molecular mechanisms by which E2F1 contributes to TGFß-mediated apoptosis and found that TGFß treatment led to the formation of a transcriptionally active E2F1-pRb-P/CAF complex on multiple TGFß pro-apoptotic target gene promoters, thereby activating their transcription. Together, our findings define a novel process of gene activation by the TGFß-E2F1 signaling axis and highlight E2F1 as a central mediator of the TGFß apoptotic program.

Calnexin-dependent regulation of tunicamycin-induced apoptosis in breast carcinoma MCF-7 cells.

The endoplasmic reticulum (ER) has evolved specific mechanisms to ensure protein folding as well as the maintenance of its own homeostasis. When these functions are not achieved, specific ER stress signals are triggered to activate either adaptive or apoptotic responses. Here, we demonstrate that MCF-7 cells are resistant to tunicamycin-induced apoptosis. We show that the expression level of the ER chaperone calnexin can directly influence tunicamycin sensitivity in this cell line. Interestingly, the expression of a calnexin lacking the chaperone domain (DeltaE) partially restores their sensitivity to tunicamycin-induced apoptosis. Indeed, we show that DeltaE acts as a scaffold molecule to allow the cleavage of Bap31 and thus generate the proapoptotic p20 fragment. Utilizing the ability of MCF-7 cells to resist tunicamycin-induced apoptosis, we have characterized a molecular mechanism by which calnexin regulates ER-stress-mediated apoptosis in a manner independent of its chaperone functions but dependent of its binding to Bap31.

Menin and TGF-beta superfamily member signaling via the Smad pathway in pituitary, parathyroid and osteoblast.

PITUITARY: Menin is a Smad3-interacting protein; inactivation of menin blocks transforming growth factor (TGF)-beta and activin signaling, antagonizing their growth-inhibitory properties in anterior pituitary cells. Menin is also required for the activin-induced inhibition of prolactin expression mediated by the Smads and the transcription factor, Pit-1. The interaction between menin and Smad3 is direct. PARATHYROID: In cultured parathyroid cells from uremic hemodialysis patients, in which the menin signaling pathways are probably still intact, menin inactivation achieved by menin antisense oligonucleotides leads to loss of TGF-beta inhibition of parathyroid cell proliferation and parathyroid hormone (PTH) secretion. Moreover, TGF-beta does not affect the proliferation and PTH production of parathyroid cells from multiple endocrine neoplasia type 1 (MEN1) patients. OSTEOBLAST: Men1-null mouse fetuses that die at day 12 or earlier have cranial/facial hypoplasias implicating menin in bone development. Menin is required for the commitment of multipotential mesenchymal stem cells into the osteoblast lineage. This is achieved by menin interacting physically and functionally with bone morphogenetic protein (BMP)-2 regulated Smads, such as Smad1 and Smad5, and the key osteoblast regulator, Runx2. These interactions are lost as the committed osteoblasts differentiate further at which time menin interacts with Smad3, mediating the negative regulation of Runx2 by TGF-beta. Menin also suppresses osteoblast maturation, partly by inhibiting the differentiation actions of JunD.

The p38 MAPK pathway is required for cell growth inhibition of human breast cancer cells in response to activin.

Activin, a member of the TGFbeta family inhibits cell growth in various target tissues. Activin interacts with a complex of two receptors that upon activation phosphorylate specific intracellular mediators, the Smad proteins. The activated Smads interact with diverse DNA binding proteins and co-activators of transcription in a cell-specific manner, thus leading to various activin biological effects. In this study, we investigated the role and mechanism of action of activin in the human breast cancer T47D cells. We found that activin treatment of T47D cells leads to a dramatic decrease in cell growth. Thus activin appears as a potent cell growth inhibitor of these breast cancer cells. We show that activin induces the Smad pathway in these cells but also activates the p38-mitogen-activated protein kinase pathway, further leading to phosphorylation of the transcription factor ATF2. Finally, specific inhibitors of the p38 kinase (SB202190, SB203580, and PD169316) but not an inactive analogue (SB202474) or the MEK-1 inhibitor PD98059 completely abolish the activin-mediated cell growth inhibition of T47D cells. Together, these results define a new role for activin in human breast cancer T47D cells and highlight a new pathway utilized by this growth factor in the mediation of its biological effects in cell growth arrest.

Inactivation of menin, a Smad3-interacting protein, blocks transforming growth factor type beta signaling.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder characterized by endocrine tumors of parathyroids, pancreatic islets, and anterior pituitary. The MEN1 gene encodes a nuclear protein called menin. In MEN1 carriers inactivating mutations give rise to a truncated product consistent with menin acting as a tumor suppressor gene. However, the role of menin in tumorigenesis and its physiological functions are not known. Here, we show that menin inactivation by antisense RNA antagonizes transforming growth factor type beta-mediated cell growth inhibition. Menin interacts with Smad3, and antisense menin suppresses transforming growth factor type beta-induced and Smad3-induced transcriptional activity by inhibiting Smad3/4-DNA binding at specific transcriptional regulatory sites. These results implicate a mechanism of tumorigenesis by menin inactivation.

Interruption of activin A autocrine regulation by antisense oligodeoxynucleotides accelerates liver tumor cell proliferation.

Administration of activin A, a member of the transforming growth factor-beta superfamily inhibits hepatocyte proliferation in vitro and reduces liver mass in vivo. However, a role of endogenous activin A in local growth modulation has not been established in any system. The aim of this study was to examine the production of activin A in the human hepatoma cell line HLF and to explore a possible autocrine role of activin as a cell growth inhibitor by blocking production of endogenous activin using antisense oligodeoxynucleotides. Administration of exogenous activin A suppressed HLF cell growth, and immunoreactive activin A was shown to be produced in the cells at confluency by Western blotting analysis. Cells were exposed to phosphorothioate-modified oligodeoxynucleotides, synthesized with antisense or randomly shuffled base sequences of activin betaA subunit messenger RNA, under serum-free conditions. Uptake of the oligodeoxynucleotides into the cells was confirmed by use of fluorescein isothiocyanate-labeled oligodeoxynucleotides. Administration of antisense oligodeoxynucleotides reduced activin A production as confirmed by both competitive PCR and Western blotting. Activin betaA antisense oligodeoxynucleotides significantly increased cell proliferation compared with controls. These findings are consistent with the existence of an autocrine role of activin A as an inhibitor of hepatocyte proliferation.

Roles of pathway-specific and inhibitory Smads in activin receptor signaling.

Activins and other members of the transforming growth factor-beta-like superfamily of growth factors transduce their signals by interacting with two types of receptor serine/threonine kinases. The Smad proteins, a new family of intracellular mediators are involved in the signaling pathways of these receptors, but the initial stages of their activation as well as their specific functions remain to be defined. We report here that the pathway-specific Smad2 and 3 can form a complex with the activin receptor in a ligand-dependent manner. This complex formation is rapid but also transient. Indeed, soon after their association with the activin receptor, Smad2 and Smad3 are released into the cytoplasm where they interact with the common partner Smad4. These Smad complexes then mediate activin-induced transcription. Finally, we show that the inhibitory Smad7 can prevent the association of the two pathway-specific Smads with the activin receptor complex, thereby blocking the activin signal.

Tail regression, apoptosis and thyroid hormone regulation of myosin heavy chain isoforms in Xenopus tadpoles.

T3 effects on myosin heavy chain gene expression were analysed in muscles undergoing different fates during metamorphosis. Muscle fate was followed by somatic gene transfer of a constitutively expressed luciferase vector. Persistent expression was found in dorsal muscle which is remodelled during metamorphosis whilst the signal disappeared in apoptosing caudal muscle. RNAse protection assay was used to follow production of myosin heavy chain isoforms: two isoforms identified as embryonic (E3 and E19) and one adult form (A7). The effects of T3 treatment were followed over 120 h. During this time frame E3 and A7 expression patterns were found to be similar in both caudal and dorsal muscles. Most notably, at 48 h E3 expression was significantly down-regulated and production of A7 significantly upregulated in both caudal and dorsal muscle. Thus T3-induced transitions in muscle gene expression are independent of muscle fate during amphibian metamorphosis.

Inhibition of tumor necrosis factor-alpha action within the CNS markedly reduces the plasma adrenocorticotropin response to peripheral local inflammation in rats.

The present study tested the hypothesis that the cytokine tumor necrosis factor-alpha (TNF-alpha) is an important CNS mediator of the hypothalamo-pituitary-adrenal (HPA) axis response to local inflammation in the rat. Recombinant murine TNF-alpha administered directly into the cerebroventricles of normal rats produced a dose-dependent increase in plasma adrenocorticotropin (ACTH) concentration. Local inflammation induced by the intramuscular injection of turpentine (50 microl/100 gm body weight) also produced an increase in plasma ACTH, peaking at 160-200 pg/ml at 7.5 hr after injection (compared with 10-30 pg/ml in controls). Intracerebroventricular pretreatment with either 5 microl of anti-TNF-alpha antiserum or 1-50 microg of soluble TNF receptor construct (rhTNFR:Fc) reduced the peak of the ACTH response to local inflammation by 62-72%. In contrast, intravenous treatment with the same doses of anti-TNF-alpha or rhTNFR:Fc had no significant effect on the ACTH response to local inflammation. Although these data indicated an action of TNF-alpha specifically within the brain, no increase in brain TNF-alpha protein (measured by bioassay) or mRNA (assessed using either in situ hybridization histochemical or semi-quantitative RT-PCR procedures) was demonstrable during the onset or peak of HPA activation produced by local inflammation. Furthermore, increased passage of TNF-alpha from blood to brain seems unlikely, because inflammation did not affect plasma TNF-alpha biological activity. Collectively these data demonstrate that TNF-alpha action within the brain is critical to the elaboration of the HPA axis response to local inflammation in the rat, but they indicate that increases in cerebral TNF-alpha synthesis are not a necessary accompaniment.

Activin and inhibin have antagonistic effects on ligand-dependent heteromerization of the type I and type II activin receptors and human erythroid differentiation.

Activins and inhibins belong to the transforming growth factor beta (TGF-beta)-like superfamily and exert their effects on a broad range of cellular targets by modulating cell differentiation and proliferation. Members of this family interact with two structurally related classes of receptors (type I and type II), both containing a serine/threonine kinase domain. When expressed alone, the type II but not the type I activin receptor can bind activin. However, the presence of a type I receptor is required for signaling. For TGF-beta1, ligand binding to the type II receptor results in the recruitment and transphosphorylation of the type I receptor. Transient overexpression of the two types of activin receptor results in ligand-independent receptor heteromerization and activation. Nevertheless, activin addition to the transfected cells increased complex formation between the two receptors, suggesting a mechanism of action similar to that observed for the TGF-beta receptor. In the present study, we generated a stable cell line, overexpressing the two types of activin receptor upon induction, in the human erythroleukemia cell line K562. We demonstrate here that activin specifically induces heteromer formation between the type I and type II receptors in a time-dependent manner. Using this stable line, we analyzed the effects of activin and inhibin on human erythroid differentiation. Our results indicate that activin signal transduction mediated through its type I and type II receptors results in an increase in the hemoglobin content of the cells and limits their proliferation. Finally, using cell lines that can be induced to overexpress ActRII and ActRIB or ActRIB only, we show that the inhibin antagonistic effects on activin-induced biological responses are mediated through a competition for the type II activin receptor but also require the presence of an inhibin-specific binding component.

Regulation of transforming growth factor beta- and activin-induced transcription by mammalian Mad proteins.

Members of the transforming growth factor beta (TGF-beta) superfamily are involved in diverse physiological activities including development, tissue repair, hormone regulation, bone formation, cell growth, and differentiation. At the cellular level, these functions are initiated by the interaction of ligands with specific transmembrane receptors with intrinsic serine/threonine kinase activity. The signaling pathway that links receptor activation to the transcriptional regulation of the target genes is largely unknown. Recent work in Drosophila and Xenopus signaling suggested that Mad (Mothers against dpp) functions downstream of the receptors of the TGF-beta family. Mammalian Mad1 has been reported to respond to bone morphogenetic protein (BMP), but not to TGF-beta or activin. We report here the cloning and functional studies of a novel mammalian Mad molecule, Mad3, as well as a rat Mad1 homologue. Overexpression of Mad3 in a variety of cells stimulated basal transcriptional activity of the TGF-beta/activin-responsive reporter construct, p3TP-Lux. Furthermore, expression of Mad3 could potentiate the TGF-beta- and activin-induced transcriptional stimulation of p3TP-Lux. By contrast, overexpression of Mad1 inhibited the basal as well as the TGF-beta/activin induced p3TP-Lux activity. These findings, therefore, support the hypothesis that Mad3 may serve as a mediator linking TGF-beta/activin receptors to transcriptional regulation.

Use of heterologous DNA-based gene transfer to follow physiological, T3-dependent regulation of myosin heavy chain genes in Xenopus tadpoles.

In vivo gene transfer and RNase protection assay were used to follow thyroid hormone (T3)-dependent regulation of myosin heavy chain (myHC) genes in Xenopus tadpole dorsal muscle. One embryonic and one adult myHC form were measured by each approach. RNase protection assay showed that T3 decreased expression of endogenous embryonic mRNA (E3), but increased adult (A7) transcripts. Gene transfer showed that T3 exerted transcriptional effects on mammalian embryonic and adult myHc promoters injected into the same muscle. The kinetics and profiles of the transcriptional responses were superimposable on endogenous responses. The results strengthen the use of in vivo approaches for determining the roles of transcription factors and cis-regulatory sequences in integrated contexts.

PTP1D is a positive regulator of the prolactin signal leading to beta-casein promoter activation.

Stimulation of the prolactin receptor (PRLR), a member of the cytokine/growth hormone receptor family, results in activation of the associated Jak2 tyrosine kinase and downstream signaling pathways. We report that PTP1D, a cytoplasmic protein tyrosine phosphatase containing two Src homology 2 (SH2) domains, physically associates with the PRLR-Jak2 complex and is tyrosine-phosphorylated upon stimulation with prolactin. The formation of the trimeric PRLR-Jak2-PTP1D complex is critical for transmission of a lactogenic signal, while PTP1D phosphorylation is necessary, but not sufficient. The dominant negative inhibitory effect of a phosphatase-deficient mutant on expression of a beta-casein promoter-controlled reporter gene is evidence for an essential role of fully functional PTP1D in the regulation of milk protein gene transcription.

Proline-rich sequence-mediated Jak2 association to the prolactin receptor is required but not sufficient for signal transduction.

The prolactin receptor (PRLR) belongs to the superfamily of cytokine/growth hormone/prolactin receptors. Members of this family do not contain a tyrosine kinase domain but are associated with cytoplasmic kinases of the Jak family. Here, we examine different mutants of the PRLR with respect to their ability to associate and activate the kinase Jak2 and the transcription factor Stat1. Moreover, using a biological assay system we are able to correlate these activities with activation of prolactin-responsive gene transcription. Our results indicate that interaction between Jak2 and PRLR requires a proline-rich sequence in the membrane proximal region of the receptor, which is conserved among the different members of the cytokine receptor superfamily. We also show that association of Jak2 with the receptor is sufficient for activation of the kinase as well as the transcription factor Stat1. Moreover, our findings indicate that association of PRLR with Jak2 is necessary but not sufficient for the transmission of a lactogenic signal. We have identified two other cytoplasmic regions of the PRLR that are required for activation of transcription. These two regions are located between boxes 1 and 2 and are in the carboxyl-terminal tail of the receptor. These sites probably involve specific interactions with other effector molecules.

A single phosphotyrosine residue of the prolactin receptor is responsible for activation of gene transcription.

Members of the cytokine/growth hormone/prolactin (PRL) receptor superfamily are associated with cytoplasmic tyrosine kinases of the Jak family. For the PRL receptor (PRLR), after PRL stimulation, both the kinase Jak2 and the receptor undergo tyrosine phosphorylation. To assess the role of tyrosine phosphorylation of the PRLR in signal transduction, several mutant forms of the PRLR in which various tyrosine residues were changed to phenylalanine were constructed and their functional properties were investigated. We identified a single tyrosine residue located at the C terminus of the PRLR to be necessary for in vivo activation of PRL-responsive gene transcription. This clearly indicates that a phosphotyrosine residue in the cytoplasmic domain of a member of the cytokine/growth hormone/PRL receptor superfamily is directly involved in signal transduction.

Expression and modulation of the parathyroid hormone (PTH)/PTH-related peptide receptor messenger ribonucleic acid in skin fibroblasts from patients with type Ib pseudohypoparathyroidism.

To explore the possibility that defects in the regulation of expression of the messenger ribonucleic acid (mRNA) coding for the PTH receptor could be involved in pseudohypoparathyroidism type Ib (PHP-Ib), PTH-induced cAMP production and PTH/PTH-related peptide (PTH-rp) receptor mRNA expression, measured using a ribonuclease protection assay, were compared in untreated and dexamethasone (dexa)-pretreated (5 x 10(-7) mol/L; 7 days) cultured skin fibroblasts from controls (n = 4) and patients with PHP-Ib (n = 6). In control fibroblasts, stimulation of cAMP production by PTH and expression of PTH/PTH-rp receptor mRNA were easily detectable and were not significantly affected by dexa pretreatment. In fibroblasts from three PHP-Ib patients demonstrating reduced PTH-induced cAMP production that was reversed by dexa, the level of basal PTH/PTH-rp receptor mRNA was also reduced, but increased to levels similar to those in control cells after dexa pretreatment. In fibroblasts from a patient with resistance to PTH not reversed by dexa, PTH/PTH-rp receptor mRNA expression was also significantly lower than that in control cells (18 +/- 13%; P < 0.001) and remained only 30 +/- 15% of that observed in control cells after dexa pretreatment (P < 0.001). In fibroblasts from two PHP-Ib patients expressing normal cAMP responsiveness to PTH before and after dexa treatment, the level of PTH/PTH-rp receptor mRNA was not different from that in control cells before or after dexa treatment. Thus, in all conditions where PTH-induced cAMP production by PHP-Ib fibroblasts was reduced, the abnormality could be explained by the reduced level of PTH/PTH-rp receptor mRNA in these cells. These results suggest that defects in the regulation of expression of the PTH/PTH-rp receptor mRNA, not structural defects in the receptor itself, explain the PTH resistance in PHP-Ib in the patients evaluated, but several different defects must exist.

JAK2 activation and cell proliferation induced by antibody-mediated prolactin receptor dimerization.

Cytokines that interact with receptors of the hematopoietin super-family have recently been reported to stimulate receptor-associated JAK tyrosine kinases, including PRL activation of JAK2. Unlike other tyrosine kinases, none of the JAK kinases has thus far been implicated in oncogenesis, and their involvement in growth signaling has not been established. Using the PRL-dependent pre-T-cell line Nb2, the present study provided a link between bivalent dimerization of a hematopoietin receptor and activation of its associated JAK kinase, and demonstrated a strong positive correlation between the mitogenic potency of a series of bivalent anti-PRL receptor antibodies and the degree of induced tyrosine phosphorylation of JAK2. Antibody bivalency was required for JAK2 phosphorylation. Monovalent anti-PRL receptor Fab fragments alone were inactive, but their activity could be partially restored by cross-linking with bivalent anti-Fab antibodies. Additional evidence for antibody-induced receptor dimerization was provided by a bell-shaped dose-response curve for the most potent receptor agonist, monoclonal antibody T6. This phenomenon is typically seen at pharmacological concentrations of bivalent ligands, when bound ligand molecules fail to adjoin a second receptor due to occupancy. The present study provided functional support for a model of PRL receptor triggering by ligand-induced receptor homodimerization and subsequent activation of the associated tyrosine kinase JAK2.

Prolactin and the immune system.

Prolactin (PRL) is involved in a wide range of physiological effects in several species and its immunoregulatory role has already been well documented. The PRL receptor has been cloned from various species. There are at least two receptor isoforms (short and long) in rats and mice, which differ only in their cytoplasmic domains, generated by alternative splicing of a single gene, although in human only the long form exists. Using the reverse transcriptase-polymerase chain reaction (RT-PCR), we detected transcripts encoding both forms of PRL receptor in all lymphoid tissues examined in human, mouse, and rat, but in mouse and rat the ratio between the two forms was variable from animal to animal. Concerning the transcript encoding the PRL itself, a clear signal was always found in human lymphocytes and occasionally in rat thymus. We also developed a quantitative PCR (Q-PCR) in order to measure the absolute number of transcripts in thymus and spleen from rats at two stages of estrous cycle. The level of expression of the two forms was about equal. Finally, we identified the tyrosine kinase JAK2, which is constitutively associated with the PRLR, using the Nb2 rat lymphoma cell line as a model system with which to study the action of PRL on cell mitogenesis. We also showed that, after stimulation by PRL, the dimerization process is a prerequisite step for the phosphorylation of the PRLR and JAK2, which represents the earliest event in the signal transduction pathway.

Prolactin-induced proliferation of Nb2 cells involves tyrosine phosphorylation of the prolactin receptor and its associated tyrosine kinase JAK2.

The interaction of prolactin with its receptor in the Nb2 cell line has been shown to induce the phosphorylation of cell-associated proteins and mitogenesis. It has been reported previously that one of these proteins, phosphorylated upon prolactin stimulation, was a tyrosine kinase. We have identified this kinase as JAK2, and demonstrate its association with the prolactin receptor. In addition, we show that the prolactin receptor itself becomes tyrosine phosphorylated upon ligand stimulation in Nb2 cells. These actions are time-dependent and occur rapidly after prolactin stimulation, with first the kinase being activated within 5 min and then the receptor being phosphorylated maximally at 20 min. Moreover, phosphorylation of both JAK2 and the receptor as well as Nb2 cell proliferation are dependent on the concentration of lactogenic hormone, resulting in a bell-shaped response curve similar to that observed in the two site model of hGH action. This indicates that early events in signal transduction as well as later events like mitogenesis and proliferation involve prolactin receptor dimerization. Together these data indicate that the prolactin receptor in Nb2 cells is associated to JAK2 and that upon ligand stimulation, and receptor dimerization, the kinase and the receptor are tyrosine-phosphorylated, which represents the first event in the process of prolactin receptor signal transduction in Nb2 cells.

Involvement of gp130/interleukin-6 receptor transducing component in interleukin-11 receptor.

The recently cloned interleukin (IL)-11 displays many biological properties in common with those reported for IL-6. In order to analyze the nature and the functionality of the IL-11 receptor we developed a proliferative assay using the human multifactor-dependent cell line TF1. We showed that a blocking monoclonal antibody GPX7 raised against the gp130/IL-6 receptor transducing subunit was also able to inhibit the IL-11-triggered TF1 line proliferation. In addition, involvement of gp130 in IL-11 signaling was demonstrated by an induction of the transducing protein phosphorylation in response to IL-11, as observed for IL-6. In contrast, the blocking monoclonal antibody B-R6, which recognized the gp80/IL-6 binding subunit failed to interfere with the IL-11 proliferative signal in the TF1 cell line. Similarly, we did not observe any competition between IL-6 and IL-11 for a putative common binding site on the cell surface. These results suggest that the IL-11 binding component is different from the gp80/IL-6 receptor. In conclusion, IL-11, along with IL-6, leukemia inhibitory factor, oncostatin M and ciliary neurotrophic factor, belongs to the same family of cytokines, using gp130 as a transducing protein.