Polycystin-1 regulates STAT activity by a dual mechanism.

Mutations in polycystin-1 (PC1) lead to autosomal-dominant polycystic kidney disease (ADPKD), a leading cause of renal failure for which no treatment is available. PC1 is an integral membrane protein, which has been implicated in the regulation of multiple signaling pathways including the JAK/STAT pathway. Here we show that membrane-anchored PC1 activates STAT3 in a JAK2-dependent manner, leading to tyrosine phosphorylation and transcriptional activity. The C-terminal cytoplasmic tail of PC1 can undergo proteolytic cleavage and nuclear translocation. Tail-cleavage abolishes the ability of PC1 to directly activate STAT3 but the cleaved PC1 tail now coactivates STAT3 in a mechanism requiring STAT phosphorylation by cytokines or growth factors. This leads to an exaggerated cytokine response. Hence, PC1 can regulate STAT activity by a dual mechanism. In ADPKD kidneys PC1 tail fragments are overexpressed, including a unique ∼15-kDa fragment (P15). STAT3 is strongly activated in cyst-lining epithelial cells in human ADPKD, and orthologous and nonorthologous polycystic mouse models. STAT3 is also activated in developing, postnatal kidneys but inactivated in adult kidneys. These results indicate that STAT3 signaling is regulated by PC1 and is a driving factor for renal epithelial proliferation during normal renal development and during cyst growth.

TRAF7 is an essential regulator of blood vessel integrity during mouse embryonic and neonatal development.

Targeted deletion of TRAF7 revealed that it is a crucial part of shear stress-responsive MEKK3-MEK5-ERK5 signaling pathway induced in endothelial cells by blood flow. Similar to Mekk3-, Mek5- or Erk5-deficient mice, Traf7-deficient embryos died in utero around midgestation due to impaired endothelium integrity. They displayed significantly lower expression of transcription factor Klf2, an essential regulator of vascular hemodynamic forces downstream of the MEKK3-MEK-ERK5 signaling pathway. In addition, deletion of Traf7 in endothelial cells of postnatal mice was associated with severe cerebral hemorrhage. Here, we show that besides MEKK3 and MEK5, TRAF7 associates with a planar cell polarity protein SCRIB. SCRIB binds with an N-terminal region of TRAF7, while MEKK3 associates with the C-terminal WD40 domain. Downregulation of TRAF7 as well as SCRIB inhibited fluid shear stress-induced phosphorylation of ERK5 in cultured endothelial cells. These findings suggest that TRAF7 and SCRIB may comprise an upstream part of the MEKK3-MEK5-ERK5 signaling pathway.

REH2 RNA helicase in kinetoplastid mitochondria: ribonucleoprotein complexes and essential motifs for unwinding and guide RNA (gRNA) binding.

Regulation of gene expression in kinetoplastid mitochondria is largely post-transcriptional and involves the orchestration of polycistronic RNA processing, 3'-terminal maturation, RNA editing, turnover, and translation; however, these processes remain poorly studied. Core editing complexes and their U-insertion/deletion activities are relatively well characterized, and a battery of ancillary factors has recently emerged. This study characterized a novel DExH-box RNA helicase, termed here REH2 (RNA editing associated helicase 2), in unique ribonucleoprotein complexes that exhibit unwinding and guide RNA binding activities, both of which required a double-stranded RNA-binding domain (dsRBD) and a functional helicase motif I of REH2. REH2 complexes and recently identified related particles share a multiprotein core but are distinguished by several differential polypeptides. Finally, REH2 associates transiently, via RNA, with editing complexes, mitochondrial ribosomes, and several ancillary factors that control editing and RNA stability. We propose that these putative higher order structures coordinate mitochondrial gene expression.

Independent effects of dietary fat and sucrose content on chondrocyte metabolism and osteoarthritis pathology in mice.

Obesity is one of the most significant risk factors for knee osteoarthritis. However, therapeutic strategies to prevent or treat obesity-associated osteoarthritis are limited because of uncertainty about the etiology of disease, particularly with regard to metabolic factors. High-fat-diet-induced obese mice have become a widely used model for testing hypotheses about how obesity increases the risk of osteoarthritis, but progress has been limited by variation in disease severity, with some reports concluding that dietary treatment alone is insufficient to induce osteoarthritis in mice. We hypothesized that increased sucrose content of typical low-fat control diets contributes to osteoarthritis pathology and thus alters outcomes when evaluating the effects of a high-fat diet. We tested this hypothesis in male C57BL/6J mice by comparing the effects of purified diets that independently varied sucrose or fat content from 6 to 26 weeks of age. Outcomes included osteoarthritis pathology, serum metabolites, and cartilage gene and protein changes associated with cellular metabolism and stress-response pathways. We found that the relative content of sucrose versus cornstarch in low-fat iso-caloric purified diets caused substantial differences in serum metabolites, joint pathology, and cartilage metabolic and stress-response pathways, despite no differences in body mass or body fat. We also found that higher dietary fat increased fatty acid metabolic enzymes in cartilage. The findings indicate that the choice of control diets should be carefully considered in mouse osteoarthritis studies. Our study also indicates that altered cartilage metabolism might be a contributing factor to how diet and obesity increase the risk of osteoarthritis.

Glucose-mediated tyrosine nitration in adipocytes: targets and consequences.

Hyperglycemia, a key factor in insulin resistance and diabetic pathology, is associated with cellular oxidative stress that promotes oxidative protein modifications. We report that protein nitration is responsive to changes in glucose concentrations in 3T3-L1 adipocytes. Alterations in the extent of tyrosine nitration as well as the cellular nitroproteome profile correlated tightly with changing glucose concentrations. The target proteins we identified are involved in fatty acid binding, cell signaling, protein folding, energy metabolism, antioxidant capacity, and membrane permeability. The nitration of adipocyte fatty acid binding protein (FABP4) at Tyr19 decreases, similar to phosphorylation, the binding of palmitic acid to the fatty acid-free protein. This potentially alters intracellular fatty acid transport, nuclear translocation of FABP4, and agonism of PPAR gamma. Our results suggest that protein tyrosine nitration may be a factor in obesity, insulin resistance, and the pathogenesis of diabetes.

Beta IV tubulin is selectively expressed by oligodendrocytes in the central nervous system.

Oligodendrocyte differentiation and myelination involve dramatic changes in cell signaling pathways, gene expression patterns, cell shape, and cytoskeletal organization. In a pilot study investigating CNS angiogenesis, oligodendrocytes were intensely labeled by antisera directed against the C-terminal of Tie-2, a 140-kDa transmembrane receptor for angiopoietin. Immunoprecipitation of rat brain proteins with Tie-2 C-terminal antisera, however, produced a single spot of approximately 55-kDa pI approximately 5 by two-dimensional (2D) electrophoresis, which was identified as beta-tubulin by mass spectrometry. Isotype-specific antibodies for beta(IV) tubulin selectively labeled oligodendrocytes. First detected in premyelinating oligodendrocytes, beta(IV) tubulin was abundant in myelinating oligodendrocyte perinuclear cytoplasm and processes extending to and along developing myelin internodes. Beta(IV) tubulin-positive MTs were diffusely distributed in oligodendrocyte perinuclear cytoplasm and not organized around the centrosome. Beta(IV) tubulin may play a role in establishing the oligodendrocyte MT network, which is essential for the transport of myelin proteins, lipids, and RNA during myelination.

The tetraspanin protein, CD9, is expressed by progenitor cells committed to oligodendrogenesis and is linked to beta1 integrin, CD81, and Tspan-2.

Previous studies identified the tetraspanin protein CD9 in myelinating oligodendrocytes. The present report extends these observations by identifying CD9 in a subpopulation of oligodendrocyte progenitor cells (OPCs) and in premyelinating oligodendrocytes in rodents. NG2-positive cells expressed CD9 in a temporal and spatial pattern during development that was consistent with CD9 expression in OPCs just prior to their differentiation into premyelinating oligodendrocytes. NG2-positive cells in mature brains were CD9-negative. CD9 expression during oligodendrocyte development in vitro supported this hypothesis, as all CD9-positive cells became O4-positive when switched to oligodendrocyte differentiating media. CD9 immunoreactivity was enriched in myelinating oligodendrocytes and their processes, and the outer aspects of myelin internodes. Immunoprecipitation of CD9 from postnatal rat cerebrum coprecipitated beta1 integrin, CD81, and Tspan-2, another tetraspanin protein recently identified in oligodendrocytes. Following surface biotinylation of oligodendrocytes in vitro, biotinylated beta1 integrin was identified in a CD9 immunoprecipitate. These data support a molecular link between surface integrins and a CD9, Tspan-2 molecular web during the differentiation of oligodendrocytes. Oligodendrocyte production and myelination appears to be normal in CD9-deficient mice. These data support the hypothesis that CD9 helps form the tetraspanin web beneath the plasma membranes of progenitor cells committed to oligodendrogenesis, but that CD9 is not essential for oligodendrogenesis and myelination.