α1A-Adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α₁-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α₁-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α_1A-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α₁-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α_1A-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α_1A-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α_1A-AR agonism.     

α(1A)-adrenergic receptor differentially regulates STAT3 phosphorylation through PKCϵ and PKCδ in myocytes

Previous studies demonstrated α?-adrenergic receptors (ARs) increase STAT3 activation in transfected and non-cardiac primary cell lines. However, the mechanism used by α?-ARs resulting in STAT3 activation is unknown. While other G-protein-coupled receptors (GPCRs) can couple to STAT3, these mechanisms demonstrate coupling through SRC, TYK, Rac, or complex formation with Gq and used only transfected cell lines. Using normal and transgenic mice containing constitutively active mutations (CAM) of the α(1A)-AR subtype, neonatal mouse myocytes and whole hearts were analyzed for the mechanism to couple to STAT3 activation. α?-ARs stimulated time-dependent increases in p-SRC, p-JAK2, and p-STAT3 in normal neonatal myocytes. Using various kinase inhibitors and siRNA, we determined that the α(1A)-AR coupled to STAT3 through distinct and unique pathways in neonatal myocytes. We found that PKC? inhibition decreased p-ERK and p-Ser STAT3 levels without affecting p-Tyr STAT3. In contrast, we found that PKCδ inhibition affected p-SRC and p-JAK2 resulting in decreased p-Tyr and p-Ser STAT3 levels. We suggest a novel α(1A)-AR mediated PKC?/ERK pathway that regulates the phosphorylation status of STAT3 at Ser-727 while PKCδ couples to SRC/JAK2 to affect Tyr-705 phosphorylation. Furthermore, this pathway has not been previously described in a GPCR system that couples to STAT3. Given cell survival and protective cardiac effects induced by PKC, STAT3 and ERK signaling, our results could explain the neuroprotective and cardiac protective pathways that are enhanced with α(1A)-AR agonism.     

Nephrin phosphorylation regulates podocyte adhesion through the PINCH-1-ILK-α-parvin complex

Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism. [BMB Reports 2013; 46(4): 230-235]     

Smurf1 protein negatively regulates interferon-γ signaling through promoting STAT1 protein ubiquitination and degradation

Interferons are important cytokines that mediate antiviral, antiproliferative, antitumor, and immunoregulatory activities. However, uncontrolled IFN signaling may lead to autoimmune diseases. Here we identified Smurf1 as a negative regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation. Smurf1 interacted with STAT1 through the WW domains of Smurf1 and the PY motif in STAT1 and catalyzed K48-linked polyubiquitination of STAT1. Interestingly, the Smurf1-mediated ubiquitination and degradation did not require STAT1 tyrosine and serine phosphorylation. Subsequently, overexpression of Smurf1 attenuated IFN-γ-mediated STAT1 activation and antiviral immune responses, whereas knockdown of Smurf1 enhanced IFN-γ-mediated STAT1 activation, expression of STAT1 target genes, and antiviral immune responses. Furthermore, IFN-γ stimulation led to enhanced expression of Smurf1. Therefore, our results demonstrate that Smurf1 is a negative feedback regulator for IFN-γ signaling by targeting STAT1 for ubiquitination and proteasomal degradation.     

The −1030/−862-linked TNF promoter single-nucleotide polymorphisms are associated with the inability to control HIV-1 viremia

Control of HIV-1 viremia and progression to AIDS has been associated with specific HLA genes. The tumor necrosis factor (TNF) and the non-classical major histocompatibility (MHC) class I chain-related A (MICA) genes are located in the genomic segment between the HLA class I and II genes and variants of both genes have been identified. We thus analyzed TNF promoter and MICA variants in a well-characterized group of HIV-1 infected individuals with different abilities to control HIV-1 viremia. In our cohort, the –1030/–862-linked TNF promoter single-nucleotide polymorphisms (SNPs), but not MICA variants, are significantly associated with lack of control of HIV-1 viremia (P=0.03). This association is independent of those HLA-B35 alleles associated with HIV-1 disease progression with which the –862 TNF SNP has previously been independently associated. Thus, non-randomly associated genes near the TNF locus are likely involved in control of HIV-1 viremia.     

The protease Omi regulates mitochondrial biogenesis through the GSK3β/PGC-1α pathway

Loss of the mitochondrial protease activity of Omi causes mitochondrial dysfunction, neurodegeneration with parkinsonian features and premature death in mnd2 (motor neuron degeneration 2) mice. However, the detailed mechanisms underlying this pathology remain largely unknown. Here, we report that Omi participates in the process of mitochondrial biogenesis, which has been linked to several neurodegenerative diseases. The mitochondrial biogenesis is deficit in mnd2 mice, evidenced by severe decreases of mitochondrial components, mitochondrial DNA and mitochondrial density. Omi cleaves glycogen synthase kinase 3β (GSK3β), a kinase promoting PPARγ coactivator-1α (PGC-1α) degradation, to regulate PGC-1α, a factor important for the mitochondrial biogenesis. In mnd2 mice, GSK3β abundance is increased and PGC-1α abundance is decreased significantly. Inhibition of GSK3β by SB216763 or overexpression of PGC-1α can restore mitochondrial biogenesis in mnd2 mice or Omi-knockdown N2a cells. Furthermore, there is a significant improvement of the movement ability of mnd2 mice after SB216763 treatment. Thus, our study identified Omi as a novel regulator of mitochondrial biogenesis, involving in Omi protease-deficient-induced neurodegeneration.Mitochondria have a vital role in neuronal death and survival.¹ As critical cellular organelles, mitochondria have highly dynamic properties, including mitochondrial fission, fusion, transport, biogenesis and degradation. The changes of those properties affect mitochondrial functions, leading to the occurrence of diseases.^{2, 3} Growing lines of evidence suggest that the mitochondrial dysfunction is involved in aging and neurodegenerative diseases, such as Alzheimer''s disease (AD), Huntington''s disease (HD) and Parkinson''s disease (PD).^{4, 5} Similar to other neurodegenerative diseases, PD is a progressive neurological disorder, which is characterized by the development of cytoplasmic aggregates known as Lewy bodies and degeneration of dopaminergic (DA) neurons in the substantia nigra of midbrain and other brain regions.⁶ In PD, dysfunction of mitochondria has been documented to be associated with disease pathogenesis in PD brains and both genetic- and toxin-induced PD animal models. In PD brains, mutations in mitochondrial DNA (mtDNA) occur more frequently than those in age-matched control; and mutations in the nuclear-encoded mtDNA polymerase-γ gene, which impair mtDNA replication and result in multiple mtDNA deletions, cause PD-like symptoms.⁵ Meanwhile, several PD-associated gene products, including α-synuclein, parkin, DJ-1, PINK1 (PTEN-induced putative kinase 1), leucine-rich repeat kinase 2, ubiquitin carboxy-terminal hydrolase L1 and Omi, have been identified to be associated with PD, and lead to mitochondrial dysfunction with changes in mitochondrial morphology, biogenesis and mitophagy in vivo and in vitro.^{5, 7, 8, 9} Besides, mitochondrial toxins, such as MPTP (1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and rotenone that inhibit complex I of the mitochondrial respiratory chain, cause clinically parkinsonian phenotype.^{10, 11}The serine protease Omi (also known as HtrA2) belongs to the high-temperature requirement factor A (HtrA) family, and was originally identified as a mammalian homolog of the Escherichia coli heat-shock-induced serine protease HtrA/DegP and DegS.¹² Omi is mainly localized in mitochondria, although a fraction of it is also found in nucleus.¹³ Omi is released from the mitochondria into the cytosol to mediate cell death by caspase-dependent or -independent pathways in response to apoptotic stimuli.^{14, 15} However, the notion that Omi is an apoptosis inducer in the central nervous system was challenged by studies of Omi-overexpressing or -deficient mice. Omi-overexpressing mice show normal development without any sign of apoptotic cell death.¹⁶ On the other hand, mnd2 (motor neuron degeneration 2) mice that harbor protease-deficient Omi S276C mutants, and Omi-knockout mice both suffer from progressive neurodegeneration, especially in striatum, and motor abnormalities similar to PD. Both mice fail to gain weight and die before postnatal day 40 due to neurodegeneration with progressive mitochondrial damage.^{17, 18, 19} Besides, mutations in the Omi gene have also been identified in PD patients.^{20, 21} Previous studies have shown that Omi has a vital role in the mitochondrial integrity, and the loss of protease activity leads to mitochondrial dysfunction, such as abnormal mitochondrial morphology and increased mtDNA mutation and deletions, increased susceptibility of mitochondrial membrane permeabilization, decreased mitochondrial membrane potential, and reduced mitochondrial density in mnd2 mice and Omi-knockout mice.^{17, 18, 22} Omi has been found to act downstream of PINK1, but parallel to parkin, in a mitochondrial stress sensing pathway to sense the different stresses, which may be defective in PD.²³ These findings suggest that the primary function of Omi is involved in neuroprotection, especially in the maintenance of mitochondrial homeostasis.^{23, 24}In this article, we identified that Omi cleaves glycogen synthase kinase 3β (GSK3β) to regulate PPARγ coactivator-1α (PGC-1α) abundance and to ensure mitochondrial biogenesis.     

Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance

Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential.