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Transforming growth factor-␤ (TGF-␤) is an important regulator of physiological connective tissue biosynthesis and plays a central role in pathological tissue fibrosis. Previous studies have established that a biologically active lipid mediator, sphingosine 1-phosphate (S1P), mimics some of the profibrotic functions of TGF-␤ through cross-activation of Smad signaling. Here we report that another product of sphingosine kinase, dihydrosphingosine 1-phosphate (dhS1P), has an opposite role in the regulation of TGF-␤ signaling. In contrast to S1P, dhS1P inhibits TGF-␤-induced Smad2/3 phosphorylation and up-regulation of collagen synthesis. The effects of dhS1P require a lipid phosphatase, PTEN, a key modulator of cell growth and survival. dhS1P stimulates phosphorylation of the C-terminal domain of PTEN and its subsequent translocation into the nucleus. We demonstrate a novel function of nuclear PTEN as a co-factor of the Smad2/3 phosphatase, PPM1A. Complex formation of PTEN with PPM1A does not require the lipid phosphatase activity but depends on phosphorylation of the serine/threonine residues located in the C-terminal domain of PTEN. Upon complex formation with PTEN, PPM1A is protected from degradation induced by the TGF-␤ signaling. Consequently, overexpression of PTEN abrogates TGF-␤-induced Smad2/3 phosphorylation. This study establishes a novel role for nuclear PTEN in the stabilization of PPM1A. PTEN-mediated cross-talk between the sphingolipid and TGF-␤ signaling pathways may play an important role in physiological and pathological TGF-␤ signaling. TGF-␤2 is a multifunctional polypeptide growth factor that regulates cell proliferation, functional differentiation, extracellular matrix (ECM) production, cell motility, and apoptosis (1). TGF-␤ signaling is initiated by ligand binding to a heteromeric complex of transmembrane serine/threonine kinases (type I and type II) and subsequent activation of transcriptional coregulators, Smad2 and Smad3 (1). Deregulated TGF-␤ signaling has been implicated in various pathological conditions, including fibrosis and cancer. Depending on the cancer stage, the TGF-␤ signaling pathway may either suppress or promote tumorigenesis (2). Recent studies suggest that TGF-␤ may also influence tumor growth through modulation of the tumor microenvironment (3). TGF-␤ is a potent inducer of the ECM and is required under physiologic conditions, such as wound repair, to induce fibroblasts to produce and contract ECM (4). On the other hand, deregulated TGF-␤ signaling has long been postulated to underlie pathologic fibrosis, but the specific mechanisms involved in this process have not been fully delineated (5). Recent in vivo and in vitro studies also suggest a novel role for the lipid and protein phosphatase, PTEN, as an inhibitor of fibrosis. Reduced levels of PTEN were found in myofibroblasts in human lung specimens of patients with idiopatic pulmonary fibrosis (6). Additional studies using animal models of fibrosis further supported the antifibrotic role of PTEN (6 -8). The specific ro...

The presence of the HLA-B35 allele has emerged as an important risk factor for the development of isolated pulmonary hypertension in patients with scleroderma, however the mechanisms underlying this association have not been fully elucidated. The goal of our study was to determine the molecular mechanisms that mediate the biological effects of HLA-B35 in endothelial cells (ECs). Our data demonstrate that HLA-B35 expression at physiological levels via adenoviral vector resulted in significantly increased endothelin-1 (ET-1) and a significantly decreased endothelial NO synthase (eNOS), mRNA, and protein levels. Furthermore, HLA-B35 greatly upregulated expression of chaperones, including heat shock proteins (HSPs) HSP70 (HSPA1A and HSPA1B) and HSP40 (DNAJB1 and DNAJB9), suggesting that HLA-B35 induces the endoplasmic reticulum (ER) stress and unfolded protein response in ECs. Examination of selected mediators of the unfolded protein response, including H chain binding protein (BiP; GRP78), C/Ebp homologous protein (CHOP; GADD153), endoplasmic reticulum oxidase, and protein disulfide isomerase has revealed a consistent increase of BiP expression levels. Accordingly, thapsigargin, a known ER stress inducer, stimulated ET-1mRNAand protein levels in ECs. This study suggests that HLA-B35 could contribute to EC dysfunction via ER stress-mediated induction of ET-1 in patients with pulmonary hypertension.

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Our group previously identified two novel genes, RFP2/LEU5 and DLEU2, within a 13q14.3 genomic region of loss seen in various malignancies. However, no specific inactivating mutations were found in these or other genes in the vicinity of the deletion, suggesting that a nonclassical tumor-suppressor mechanism may be involved. Here, we present data showing that the DLEU2 gene encodes a putative noncoding antisense RNA, with one exon directly overlapping the first exon of the RFP2/LEU5 gene in the opposite orientation. In addition, the RFP2/LEU5 transcript can be alternatively spliced to produce either several monocistronic transcripts or a putative bicistronic transcript encoding two separate open-reading frames, adding to the complexity of the locus. The finding that these gene structures are conserved in the mouse, including the putative bicistronic RFP2/LEU5 transcript as well as the antisense relationship with DLEU2, further underlines the significance of this unusual organization and suggests a biological function for DLEU2 in the regulation of RFP2/LEU5.