Prostaglandin F2α inhibits adipogenesis via an autocrine-mediated interleukin-11/glycoprotein 130/STAT1-dependent signaling cascade.

Prostaglandin F2α (PGF2α) is a potent inhibitor of adipocyte differentiation in vitro, that has also recently been implicated in the regulation of the adipogenic process in vivo, by opposing adipose tissue accretion and the subsequent development of obesity and its attendant metabolic consequences. In previous studies, we have demonstrated that PGF2α inhibits adipocyte differentiation by means of a calcium-dependent signaling pathway that is critically dependent upon the activity of the calcineurin phosphatase. In the current study, we have now extended these findings to further elucidate the mechanism by which the PGF2α/calcineurin-pathway inhibits the adipogenic process. We now report that the IL-11 cytokine, a member of the gp130 cytokine co-receptor-related family, is a downstream transcriptional target of this pathway in 3T3-L1 preadipocytes and is actively secreted in differentiating cells in response to PGF2α stimulation. Using a combined shRNA and dominant-negative receptor mutant approach, we provide evidence that IL-11/gp130-signaling is required to mediate the inhibitory effects of PGF2α on adipogenesis. Moreover, by taking advantage of a well-characterized panel of chimeric gp130 mutant receptors, we demonstrate that gp130 signaling is sufficient to inhibit adipocyte differentiation and specifically requires the activation of the STAT1 transcription factor. Conversely, we find that depleting endogenous STAT1 levels rescues adipogenesis in the presence of both IL-11/gp130 signaling and PGF2α. Collectively, our findings support a model in which PGF2α inhibits adipocyte differentiation by means of an IL-11 mediated autocrine negative feedback loop, that acts via gp130 to block adipogenesis through the essential actions of the STAT1 transcription factor.

Deregulated NFATc1 activity transforms murine fibroblasts via an autocrine growth factor-mediated Stat3-dependent pathway.

The nuclear factor of activated T cells (NFAT) family of transcription factors has recently been implicated with a role in tumorigenesis. Forced expression of a constitutively active NFATc1 mutant (caNFATc1) has been shown to transform immortalized murine fibroblasts in vitro, while constitutive activation of the NFAT-signaling pathway has been found in a number of human cancers, where it has been shown to contribute towards various aspects of the tumor phenotype. Here we have investigated the molecular mechanisms underlying the oncogenic potential of deregulated NFAT activity. We now show that ectopic expression of caNFATc1 in murine 3T3-L1 fibroblasts induces the secretion of an autocrine factor(s) that is sufficient to promote the transformed phenotype. We further demonstrate that this NFATc1-induced autocrine factor(s) specifically induces the tyrosine phosphorylation of the Stat3 transcription factor via a JAK kinase-dependent pathway. Interestingly, this effect of sustained NFAT signaling on the autocrine growth factor-mediated activation of Stat3 is not restricted to murine fibroblasts, but is also observed in the PANC-1 and MCF10A human cell lines. Most importantly, we find that the shRNA-mediated depletion of endogenous Stat3 significantly attenuates the ability of caNFATc1 to transform 3T3-L1 fibroblasts. Taken together, our results afford significant new insights into the molecular mechanisms underlying the oncogenic potential of deregulated NFATc1 activity by demonstrating that constitutive NFATc1 activity transforms cells via an autocrine factor-mediated pathway that is critically dependent upon the activity of the Stat3 transcription factor.

Prostaglandin F2alpha induces the normoxic activation of the hypoxia-inducible factor-1 transcription factor in differentiating 3T3-L1 preadipocytes: Potential role in the regulation of adipogenesis.

Prostaglandin F2alpha (PGF2alpha) is a potent paracrine inhibitor of adipocyte differentiation. Here we show that treatment of differentiating 3T3-L1 preadipocytes with PGF2alpha induces the expression of DEC1, a transcriptional repressor that has previously been implicated in the inhibition of adipogenesis in response to hypoxia as a downstream effector of the hypoxia-inducible factor-1 (HIF-1) transcription factor. Surprisingly, despite performing our experiments under normal ambient oxygen conditions, we find that treatment of differentiating 3T3-L1 preadipocytes with PGF2alpha also results in the marked activation of HIF-1, as measured by an increase in the accumulation of the HIF-1alpha regulatory subunit. However, unlike the effects of hypoxia, this PGF2alpha-induced normoxic increase in HIF-1alpha is not mediated by an increase in the stability of the HIF-1alpha polypeptide, rather we find that PGF2alpha selectively increases the expression of the alternatively spliced HIF-1alpha I.1 mRNA isoform. Significantly, we demonstrate that the shRNA-mediated knockdown of endogenous HIF-1alpha expression attenuates the PGF2alpha-induced expression of DEC1, overcomes the inhibitory effects of PGF2alpha on the expression of proadipogenic transcription factors C/EBPalpha and PPARgamma and partially rescues the PGF2alpha-induced inhibition of adipogenesis. Taken together, these results indicate that PGF2alpha promotes the activation of the HIF-1 transcription factor pathway under normal oxygen conditions, and highlight a potential role for the normoxic activation of the HIF-1/DEC1-pathway in mediating the inhibitory effects of PGF2alpha on adipocyte differentiation.

Modulation of NFAT-dependent gene expression by the RhoA signaling pathway in T cells.

We have reported previously that p115Rho guanine nucleotide exchange factor, its upstream activator Galpha13, and its effector RhoA are able to inhibit HIV-1 replication. Here, we show that RhoA is able to inhibit HIV-1 gene expression through the NFAT-binding site in the HIV long-terminal repeat. Constitutively active NFAT counteracts the inhibitory activity of RhoA, and inhibition of NFAT activation also inhibits HIV-1 gene expression. We have shown further that RhoA inhibits NFAT-dependent transcription and IL-2 production in human T cells. RhoA does not inhibit nuclear localization of NFAT but rather, inhibits its transcriptional activity. In addition, RhoA decreases the level of acetylated histone H3, but not NFAT occupancy, at the IL-2 promoter. These data suggest that activation of RhoA can modulate IL-2 gene expression by inhibiting the transcriptional activity of NFAT and chromatin structure at the IL-2 promoter during T cell activation.

Prostaglandin F2alpha inhibits adipocyte differentiation via a G alpha q-calcium-calcineurin-dependent signaling pathway.

Prostaglandin F2alpha (PGF2alpha) is a potent physiological inhibitor of adipocyte differentiation, however the specific signaling pathways and molecular mechanisms involved in mediating its anti-adipogenic effects are not well understood. In the current study, we now provide evidence that PGF2alpha inhibits adipocyte differentiation via a signaling pathway that requires heterotrimeric G-protein G alpha q subunits, the elevation of the intracellular calcium concentration ([Ca2+]i), and the activation of the Ca2+/calmodulin-regulated serine/threonine phosphatase calcineurin. We show that while this pathway acts to inhibit an early step in the adipogenic cascade, it does not interfere with the initial mitotic clonal expansion phase of adipogenesis, nor does it affect either the expression, DNA binding activity or differentiation-induced phosphorylation of the early transcription factor C/EBPbeta. Instead, we find that PGF2alpha inhibits adipocyte differentiation via a calcineurin-dependent mechanism that acts to prevent the expression of the critical pro-adipogenic transcription factors PPARgamma and C/EBPalpha. Furthermore, we demonstrate that the inhibitory effects of PGF2alpha on both the expression of PPARgamma and C/EBPalpha and subsequent adipogenesis can be attenuated by treatment of preadipocytes with the histone deacetylase (HDAC) inhibitor trichostatin A. Taken together, these results indicate that PGF2alpha inhibits adipocyte differentiation via a G alpha q-Ca2+-calcineurin-dependent signaling pathway that acts to block expression of PPARgamma and C/EBPalpha by a mechanism that appears to involves an HDAC-sensitive step.

The calcineurin/nuclear factor of activated T cells signaling pathway regulates osteoclastogenesis in RAW264.7 cells.

Although best known for its role in T lymphocyte activation, the calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway is also known to be involved in a wide range of other biological responses in a variety of different cell types. Here we have investigated the role of the calcineurin/NFAT signaling pathway in the regulation of osteoclast differentiation. Osteoclasts are bone-resorbing multinucleated cells that are derived from the monocyte/macrophage cell lineage after stimulation with a member of the tumor necrosis factor family of ligands known as receptor activator of nuclear factor-kappaB ligand (RANKL). We now report that inhibition of calcineurin with either the immunosuppressant drugs cyclosporin A and FK506, or the retrovirally mediated ectopic expression of a specific calcineurin inhibitory peptide, all potently inhibit the RANKL-induced differentiation of the RAW264.7 monocyte/macrophage cell line into mature multinucleated osteoclasts. In addition, we find that NFAT family members are expressed in RAW264.7 cells and that their expression is up-regulated in response to RANKL stimulation. Most importantly, we find that ectopic expression of a constitutively active, calcineurin-independent NFATc1 mutant in RAW264.7 cells is sufficient to induce these cells to express an osteoclast-specific pattern of gene expression and differentiate into morphologically distinct, multinucleated osteoclasts capable of inducing the resorption of a physiological mineralized matrix substrate. Taken together, these data define calcineurin as an essential downstream effector of the RANKL-induced signal transduction pathway leading toward the induction of osteoclast differentiation and furthermore, indicate that the activation of the NFATc1 transcription factor is sufficient to initiate a genetic program that results in the specification of the mature functional osteoclast cell phenotype.

Infection with Theiler's murine encephalomyelitis virus directly induces proinflammatory cytokines in primary astrocytes via NF-kappaB activation: potential role for the initiation of demyelinating disease.

Theiler's virus infection in the central nervous system (CNS) induces a demyelinating disease very similar to human multiple sclerosis. We have assessed cytokine gene activation upon Theiler's murine encephalomyelitis virus (TMEV) infection and potential mechanisms in order to delineate the early events in viral infection that lead to immune-mediated demyelinating disease. Infection of SJL/J primary astrocyte cultures induces selective proinflammatory cytokine genes (interleukin-12p40 [IL-12p40], IL-1, IL-6, tumor necrosis factor alpha, and beta interferon [IFN-beta]) important in the innate immune response to infection. We find that TMEV-induced cytokine gene expression is mediated by the NF-kappaB pathway based on the early nuclear NF-kappaB translocation and suppression of cytokine activation in the presence of specific inhibitors of the NF-kappaB pathway. Further studies show this to be partly independent of dsRNA-dependent protein kinase (PKR) and IFN-alpha/beta pathways. Altogether, these results demonstrate that infection of astrocytes and other CNS-resident cells by TMEV provides the early NF-kappaB-mediated signals that directly activate various proinflammatory cytokine genes involved in the initiation and amplification of inflammatory responses in the CNS known to be critical for the development of immune-mediated demyelination.

Induction of FucT-VII by the Ras/MAP kinase cascade in Jurkat T cells.

Induction of the alpha1,3-fucosyltransferase FucT-VII in T lymphocytes is crucial for selectin ligand formation, but the signaling and transcriptional pathways that govern FucT-VII expression are unknown. Here, using a novel, highly phorbol myristate acetate (PMA)-responsive variant of the Jurkat T-cell line, we identify Ras and downstream mitogen-activated protein (MAP) kinase pathways as essential mediators of FucT-VII gene expression. PMA induced FucT-VII in only a subset of treated cells, similar to expression of FucT-VII in normal activated CD4 T cells. Introduction of constitutively active Ras or Raf by recombinant retroviruses induced FucT-VII expression only in that subset of cells expressing the highest levels of Ras, suggesting that induction of FucT-VII required a critical threshhold of Ras signaling. Both PMA treatment and introduction of active Ras led to rolling on E-selectin. Pharmacologic inhibition studies confirmed the involvement of the classic Ras-Raf-MEK-extracellular signal-regulated kinase (Ras-Raf-MEK-ERK) pathway in FucT-VII induction by PMA, Ras, and Raf. These studies also revealed a second, Ras-induced, Raf-1-independent pathway that participated in induction of FucT-VII. Strong activation of Ras represents a major pathway for induction of FucT-VII gene expression in T cells.

A constitutively active NFATc1 mutant induces a transformed phenotype in 3T3-L1 fibroblasts.

The calcineurin/nuclear factor of activated T cells (NFAT) signaling pathway is best known for its role in T lymphocyte activation. However, it has become increasingly apparent that this signaling pathway is also involved in the regulation of cell growth and development in a wide variety of different tissues and cell types. Here we have investigated the effects of sustained NFATc1 signaling on the growth and differentiation of the murine 3T3-L1 preadipocyte cell line. Remarkably, we find that expression of a constitutively active NFATc1 mutant (caNFATc1) in these immortalized cells inhibits their differentiation into mature adipocytes and causes them to adopt a transformed cell phenotype, including loss of contact-mediated growth inhibition, reduced serum growth requirements, protection from growth factor withdrawal-induced apoptosis, and formation of colonies in semisolid media. Furthermore, we find that caNFATc1-expressing cells acquire growth factor autonomy and are able to proliferate even in the complete absence of serum. We provide evidence that this growth factor independence is caused by the NFATc1-dependent production of a soluble heat-labile autocrine factor that is capable of promoting the growth and survival of wild type 3T3-L1 cells as well as potently inhibiting their differentiation into mature adipocytes. Finally, we demonstrate that cells expressing caNFATc1 form tumors in nude mice. Taken together, these results indicate that deregulated NFATc1 activity is able to induce the immortalized 3T3-L1 preadipocyte cell line to acquire the well established hallmarks of cellular transformation and thereby provide direct evidence for the oncogenic potential of the NFATc1 transcription factor.

Calcineurin mediates the calcium-dependent inhibition of adipocyte differentiation in 3T3-L1 cells.

Recent studies have revealed that the calcium-dependent serine/threonine phosphatase calcineurin mediates the effects of intracellular calcium in many different cell types. In this study we investigated the role of calcineurin in the regulation of adipocyte differentiation. We found that the specific calcineurin inhibitors cyclosporin A and FK506 overcame the antiadipogenic effect of calcium ionophore on the differentiation of 3T3-L1 preadipocytes. This finding suggests that calcineurin is responsible for mediating the previously documented Ca(2+)-dependent inhibition of adipogenesis. We further demonstrate that the expression of a constitutively active calcineurin mutant potently inhibits the ability of 3T3-L1 cells to undergo adipocyte differentiation by preventing expression of the proadipogenic transcription factors peroxisome proliferator-activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein alpha (C/EBPalpha). This calcineurin-mediated block in adipocyte differentiation is rescued by ectopic expression of PPARgamma1. Finally, we demonstrate that inhibition of endogenous calcineurin activity with either FK506 or a specific calcineurin inhibitory peptide enhances differentiation of 3T3-L1 cells in response to suboptimal adipogenic stimuli, suggesting that endogenous calcineurin activity normally sets a signaling threshold that antagonizes efficient adipocyte differentiation. Collectively, these data indicate that calcineurin acts as a Ca(2+)-dependent molecular switch that negatively regulates commitment to adipocyte differentiation by preventing the expression of critical proadipogenic transcription factors.

Differential abilities of central nervous system resident endothelial cells and astrocytes to serve as inducible antigen-presenting cells.

Microglial cells and astrocytes are capable of processing and presenting antigens for efficient activation of T cells. However, the antigen-presenting function and role of cerebrovascular endothelial cells (CVEs) in central nervous system inflammatory responses remain controversial. We compared the expression of necessary accessory molecules and the functional antigen-presenting capacity of cloned SJL/J CVEs and primary astrocytes in response to the pro-inflammatory cytokines interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha). Astrocytes and CVEs up-regulated major histocompatibility complex (MHC) class II, and primarily B7-1 as opposed to B7-2, in response to IFN-gamma. TNF-alpha inhibited the IFN-gamma-induced up-regulation of MHC class II on CVEs correlating to a decrease in the mRNA for the class II transactivator (CIITA), whereas CIITA expression in astrocytes was unaffected. Unlike astrocytes, CVEs did not elicit significant MHC class II-restricted T-cell responses. Furthermore, we have found that CVE monolayers are altered following T-cell contact, implicating CVE/T-cell contact in the breakdown of the blood-brain barrier during neuro-inflammatory responses.

Sustained NFAT signaling promotes a Th1-like pattern of gene expression in primary murine CD4+ T cells.

T cell activation is known to be critically regulated by the extent and duration of TCR-induced signaling pathways. The NFAT family of transcription factors is believed to play an important role in coupling these quantitative differences in TCR-induced signaling events into changes in gene expression. In this study we have specifically investigated the effects of sustained NFAT signaling on T cell activation by introducing a constitutively active mutant version of NFATc1 (caNFATc1) into primary murine CD4(+) T cells and examining its effects on gene expression. We now report that ectopic expression of caNFATc1 partially mimics TCR signaling, resulting in enhanced expression of CD25 and CD40 ligand and down-regulation of CD62L. More importantly, we find that expression of caNFATc1 in T cells maintained under either nonpolarizing or Th1-skewing conditions leads to a marked selective increase in the number of cells expressing the prototypical Th1 cytokine, IFN-gamma. Furthermore, when expressed in Th2-skewed cells, caNFATc1 appears to attenuate Th2 differentiation by decreasing production of IL-4 and promoting the expression of IFN-gamma. Finally, we find that caNFATc1 enhances expression of functional P-selectin glycoprotein ligand-1, up-regulates Fas ligand expression, and increases susceptibility to activation-induced cell death, cellular traits that are preferentially associated with Th1 effector cells. Taken together, these results suggest that sustained NFAT signaling, mediated by ectopic expression of caNFATc1, acts to promote a Th1-like pattern of gene expression and thereby serves to highlight the important relationship between the degree of NFAT signaling and the qualitative pattern of gene expression induced during T cell activation.