Kinases Mst1 and Mst2 positively regulate phagocytic induction of reactive oxygen species and bactericidal activity.

Mitochondria need to be juxtaposed to phagosomes for the synergistic production of ample reactive oxygen species (ROS) in phagocytes to kill pathogens. However, how phagosomes transmit signals to recruit mitochondria has remained unclear. Here we found that the kinases Mst1 and Mst2 functioned to control ROS production by regulating mitochondrial trafficking and mitochondrion-phagosome juxtaposition. Mst1 and Mst2 activated the GTPase Rac to promote Toll-like receptor (TLR)-triggered assembly of the TRAF6-ECSIT complex that is required for the recruitment of mitochondria to phagosomes. Inactive forms of Rac, including the human Rac2(D57N) mutant, disrupted the TRAF6-ECSIT complex by sequestering TRAF6 and substantially diminished ROS production and enhanced susceptibility to bacterial infection. Our findings demonstrate that the TLR-Mst1-Mst2-Rac signaling axis is critical for effective phagosome-mitochondrion function and bactericidal activity.

Repeated stress to the skin amplifies neutrophil infiltration in a keratin 17- and PKCα-dependent manner.

Neutrophils are the first immune cells to reach inflamed sites and contribute to the pathogenesis of chronic inflammatory skin diseases. Yet, little is known about the pattern of neutrophil infiltration in inflamed skin in vivo and the mechanisms mediating their recruitment. Here, we provide insight into the dynamics of neutrophil infiltration in skin in response to acute or repeated inflammatory stress, highlighting a novel keratinocyte- and keratin 17 (K17)-dependent mechanism that regulates neutrophil recruitment to inflamed skin. We used the phorbol ester TPA and UVB, alone or in combination, to induce sterile inflammation in mouse skin. A single TPA treatment results in a neutrophil influx in the dermis that peaks at 12 h and resolves within 24 h. A subsequent TPA treatment or a UVB challenge, when applied 24 h but not 48 h later, accelerates, amplifies, and prolongs neutrophil infiltration. This transient amplification response (TAR) is mediated by local signals in inflamed skin, can be recapitulated in ex vivo culture, and involves the K17-dependent sustainment of protein kinase Cα (PKCα) activity and release of chemoattractants by stressed keratinocytes. K17 binds RACK1, a scaffold protein essential for PKCα activity. The N-terminal head domain of K17 is crucial for its association with RACK1 and regulation of PKCα activity. Analysis of RNAseq data reveals a signature consistent with TAR and PKCα activation in inflammatory skin diseases. These findings uncover a novel, keratin-dependent mechanism that amplifies neutrophil recruitment in skin under stress, with direct implications for inflammatory skin disorders.

Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/ß-catenin signaling by stabilization of protein kinase Cα.

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/ß-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased ß-catenin and downregulated glycogen synthase kinase-3ß (GSK-3ß) protein levels and its activation. However, ß-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3ß was significantly increased after NDRG1 overexpression, suggesting a GSK-3ß-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Cα (PKCα), with PKCα silencing preventing ß-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing ß-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in ß-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCα, NDRG1, and ß-catenin was identified, with the formation of a potential metabolon that promotes the latter ß-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in ß-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased ß-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCα and inhibit WNT signaling.

Abnormal α-Synuclein Aggregates Cause Synaptic- and Microcircuit-Specific Deficits in the Retinal Rod Pathway.

α-Synuclein (α-Syn) is a key determinator of Parkinson disease (PD) pathology, but synapse and microcircuit pathologies in the retina underlying visual dysfunction are poorly understood. Herein, histochemical and ultrastructural analyses and ophthalmologic measurements in old transgenic M83 PD model (mice aged 16 to 18 months) indicated that abnormal α-Syn aggregation in the outer plexiform layer (OPL) was associated with degeneration in the C-terminal binding protein 2 (CtBP2)+ ribbon synapses of photoreceptor terminals and protein kinase C alpha (PKCα)+ rod bipolar cell terminals, whereas α-Syn aggregates in the inner retina correlated with the reduction and degeneration of tyrosine hydroxylase- and parvalbumin-positive amacrine cells. Phosphorylated Ser129 α-synuclein expression was strikingly restricted in the OPL, with the most severe degenerations in the entire retina, including mitochondrial degeneration and loss of ribbon synapses in 16- to 18-month-old mice. These synapse- and microcircuit-specific deficits of the rod pathway at the CtBP2+ rod terminals and PKCα+ rod bipolar and amacrine cells were associated with attenuated a- and b-wave amplitudes and oscillatory potentials on the electroretinogram. They were also associated with the impairment of visual functions, including reduced contrast sensitivity and impairment of the middle range of spatial frequencies. Collectively, these findings demonstrate that α-Syn aggregates cause the synapse- and microcircuit-specific deficits of the rod pathway and the most severe damage to the OPL, providing the retinal synaptic and microcircuit basis for visual dysfunctions in PD.

Arterial Smooth Muscle Cell AKAP150 Mediates Exercise-Induced Repression of CaV1.2 Channel Function in Cerebral Arteries of Hypertensive Rats.

BACKGROUND: Hypertension is a major, prevalent risk factor for the development and progression of cerebrovascular disease. Regular exercise has been recommended as an excellent choice for the large population of individuals with mild-to-moderate elevations in blood pressure, but the mechanisms that underlie its vascular-protective and antihypertensive effects remain unknown. Here, we describe a mechanism by which myocyte AKAP150 (A-kinase anchoring protein 150) inhibition induced by exercise training alleviates voltage-dependent L-type Ca2+ channel (CaV1.2) activity and restores cerebral arterial function in hypertension. METHODS: Spontaneously hypertensive rats and newly generated smooth muscle-specific AKAP150 knockin mice were used to assess the role of myocyte AKAP150/CaV1.2 channel in regulating cerebral artery function after exercise intervention. RESULTS: Activation of the AKAP150/PKCα (protein kinase Cα) signaling increased CaV1.2 activity and Ca2+ influx of cerebral arterial myocyte, thus enhancing vascular tone in spontaneously hypertensive rats. Smooth muscle-specific AKAP150 knockin mice were hypertensive with higher CaV1.2 channel activity and increased vascular tone. Furthermore, treatment of Ang II (angiotensin II) resulted in a more pronounced increase in blood pressure in smooth muscle-specific AKAP150 knockin mice. Exercise training significantly reduced arterial myocyte AKAP150 expression and alleviated CaV1.2 channel activity, thus restoring cerebral arterial function in spontaneously hypertensive rats and smooth muscle-specific AKAP150 knockin mice. AT1R (AT1 receptor) and AKAP150 were interacted closely in arterial myocytes. Exercise decreased the circulating Ang II and Ang II-involved AT1R-AKAP150 association in myocytes of hypertension. CONCLUSIONS: The current study demonstrates that aerobic exercise ameliorates CaV1.2 channel function via inhibiting myocyte AKAP150, which contributes to reduced cerebral arterial tone in hypertension.

Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes by PKCα-associated signalling under cholesterol stimulation.

BACKGROUND AND AIMS: The secretion of bile salts transported by the bile salt export pump (BSEP) is the primary driving force for the generation of bile flow; thus, it is closely related to the formation of cholesterol stones. Caveolin-1 (Cav-1), an essential player in cell signalling and endocytosis, is known to co-localize with cholesterol-rich membrane domains. This study illustrates the role of Cav-1 and BSEP in cholesterol stone formation. METHODS: Adult male C57BL/6 mice were used as an animal model. HepG2 cells were cultured under different cholesterol concentrations and BSEP, Cav-1, p-PKCα and Hax-1 expression levels were determined via Western blotting. Expression levels of BSEP and Cav-1 mRNA were detected using real-time PCR. Immunofluorescence and immunoprecipitation assays were performed to study BSEP and Hax-1 distribution. Finally, an ATPase activity assay was performed to detect BSEP transport activity under different cholesterol concentrations in cells. RESULTS: Under low-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels significantly increased, PKCα phosphorylation significantly decreased, BSEP binding capacity to Hax-1 weakened, and BSEP function increased. Under high-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels decreased, PKCα phosphorylation increased, BSEP binding capacity to Hax-1 rose, and BSEP function decreased. CONCLUSION: Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes via PKCα-associated signalling under cholesterol stimulation.

Crosstalk between protein kinase C α and transforming growth factor ß signaling mediated by Runx2 in intestinal epithelial cells.

Tight coordination of growth regulatory signaling is required for intestinal epithelial homeostasis. Protein kinase C α (PKCα) and transforming growth factor ß (TGFß) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFß signaling. RNA-Seq analysis of nontransformed intestinal crypt-like cells and colorectal cancer cells identified TGFß receptor 1 (TGFßR1) as a target of PKCα signaling. RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFßR1 mRNA and protein expression in these cells. Effects on TGFßR1 were dependent on Ras-extracellular signal-regulated kinase 1/2 (ERK) signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFßR1 transcription, and Runx2 was identified as an essential mediator of the effect. PKCα promoted ERK-mediated activating phosphorylation of Runx2, which preceded transcriptional activation of the TGFßR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCα→ERK→Runx2→TGFßR1 signaling axis. In further support of a link between PKCα and TGFß signaling, PKCα knockdown reduced the ability of TGFß to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFßR1 decreased PKCα-induced upregulation of p21Cip1 and p27Kip1 in intestinal cells. The physiological relevance of these findings is also supported by The Cancer Genome Atlas data showing correlation between PKCα, Runx2, and TGFßR1 mRNA expression in human colorectal cancer. PKCα also regulated TGFßR1 in endometrial cancer cells, and PKCα, Runx2, and TGFßR1 expression correlates in uterine tumors, indicating that crosstalk between PKCα and TGFß signaling may be a common mechanism in diverse epithelial tissues.

Gi/o GPCRs drive the formation of actin-rich tunneling nanotubes in cancer cells via a Gßγ/PKCα/FARP1/Cdc42 axis.

The functional association between stimulation of G-protein-coupled receptors (GPCRs) by eicosanoids and actin cytoskeleton reorganization remains largely unexplored. Using a model of human adrenocortical cancer cells, here we established that activation of the GPCR OXER1 by its natural agonist, the eicosanoid 5-oxo-eicosatetraenoic acid, leads to the formation of filopodia-like elongated projections connecting adjacent cells, known as tunneling nanotube (TNT)-like structures. This effect is reduced by pertussis toxin and GUE1654, a biased antagonist for the Gßγ pathway downstream of OXER1 activation. We also observed pertussis toxin-dependent TNT biogenesis in response to lysophosphatidic acid, indicative of a general response driven by Gi/o-coupled GPCRs. TNT generation by either 5-oxo-eicosatetraenoic acid or lysophosphatidic acid is partially dependent on the transactivation of the epidermal growth factor receptor and impaired by phosphoinositide 3-kinase inhibition. Subsequent signaling analysis reveals a strict requirement of phospholipase C ß3 and its downstream effector protein kinase Cα. Consistent with the established role of Rho small GTPases in the formation of actin-rich projecting structures, we identified the phosphoinositide 3-kinase-regulated guanine nucleotide exchange factor FARP1 as a GPCR effector essential for TNT formation, acting via Cdc42. Altogether, our study pioneers a link between Gi/o-coupled GPCRs and TNT development and sheds light into the intricate signaling pathways governing the generation of specialized actin-rich elongated structures in response to bioactive signaling lipids.

Angiotensin receptors and α1B-adrenergic receptors regulate native IK(ACh) and phosphorylation-deficient GIRK4 (S418A) channels through different PKC isoforms.

Signaling of G protein-activated inwardly rectifying K+ (GIRK) channels is an important mechanism of the parasympathetic regulation of the heart rate and cardiac excitability. GIRK channels are inhibited during stimulation of Gq-coupled receptors (GqPCRs) by depletion of phosphatidyl-4,5-bisphosphate (PIP2) and/or channel phosphorylation by protein kinase C (PKC). The GqPCR-dependent modulation of GIRK currents in terms of specific PKC isoform activation was analyzed in voltage-clamp experiments in rat atrial myocytes and in CHO or HEK 293 cells. By using specific PKC inhibitors, we identified the receptor-activated PKC isoforms that contribute to phenylephrine- and angiotensin-induced GIRK channel inhibition. We demonstrate that the cPKC isoform PKCα significantly contributes to GIRK inhibition during stimulation of wildtype α1B-adrenergic receptors (α1B-ARs). Deletion of the α1B-AR serine residues S396 and S400 results in a preferential regulation of GIRK activity by PKCß. As a novel finding, we report that the AT1-receptor-induced GIRK inhibition depends on the activation of the nPKC isoform PKCε whereas PKCα and PKCß do not mainly participate in the angiotensin-mediated GIRK reduction. Expression of the dominant negative (DN) PKCε prolonged the onset of GIRK inhibition and significantly reduced AT1-R desensitization, indicating that PKCε regulates both GIRK channel activity and the strength of the receptor signal via a negative feedback mechanism. The serine residue S418 represents an important phosphorylation site for PKCε in the GIRK4 subunit. To analyze the functional impact of this PKC phosphorylation site for receptor-specific GIRK channel modulation, we monitored the activity of a phosphorylation-deficient (GIRK4 (S418A)) GIRK4 channel mutant during stimulation of α1B-ARs or AT1-receptors. Mutation of S418 did not impede α1B-AR-mediated GIRK inhibition, suggesting that S418 within the GIRK4 subunit is not subject to PKCα-induced phosphorylation. Furthermore, activation of angiotensin receptors induced pronounced GIRK4 (S418A) channel inhibition, excluding that this phosphorylation site contributes to the AT1-R-induced GIRK reduction. Instead, phosphorylation of S418 has a facilitative effect on GIRK activity that was abolished in the GIRK4 (S418A) mutant. To summarize, the present study shows that the receptor-dependent regulation of atrial GIRK channels is attributed to the GqPCR-specific activation of different PKC isoforms. Receptor-specific activated PKC isoforms target distinct phosphorylation sites within the GIRK4 subunit, resulting in differential regulation of GIRK channel activity with either facilitative or inhibitory effects on GIRK currents.

Hepatitis C Virus Disrupts Annexin 5-Mediated Occludin Integrity through Downregulation of Protein Kinase Cα (PKCα) and PKCη Expression, Thereby Promoting Viral Propagation.

Annexins (ANXs) comprise a family of calcium- and phospholipid-binding proteins and are implicated in the hepatitis C virus (HCV) life cycle. Here, we demonstrate a novel role of ANX5 in the HCV life cycle. Comparative analysis by quantitative PCR in human hepatoma cells revealed that ANX2, ANX4, and ANX5 were highly expressed among the ANX family proteins. Knockdown of ANX5 mRNA resulted in marked enhancement of HCV RNA replication but had no effect on either HCV translation or assembly. Using the HCV pseudoparticle (HCVpp) system, we observed enhancement of HCVpp infectivity in ANX5 knockdown Huh-7OK1 cells, suggesting that ANX5 is involved in suppression of HCV entry. Additionally, we observed that subcellular localizations of tight-junction proteins, such as claudin 1 (CLDN1) and occludin (OCLN), were disrupted in the ANX5 knockdown cells. It was reported that HCV infection was facilitated by disruption of OCLN distribution and that proper distribution of OCLN was regulated by its phosphorylation. Knockdown of ANX5 resulted in a decrease of OCLN phosphorylation, thereby disrupting OCLN distribution and HCV infection. Further analysis revealed that protein kinase C (PKC) isoforms, including PKCα and PKCη, play important roles in the regulation of ANX5-mediated phosphorylation and distribution of OCLN and in the restriction of HCV infection. HCV infection reduced OCLN phosphorylation through the downregulation of PKCα and PKCη expression. Taken together, these results suggest that ANX5, PKCα, and PKCη contribute to restriction of HCV infection by regulating OCLN integrity. We propose a model that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting HCV propagation. IMPORTANCE Host cells have evolved host defense machinery to restrict viral infection. However, viruses have evolved counteracting strategies to achieve their infection. In the present study, we obtained results suggesting that ANX5 and PKC isoforms, including PKCα and PKCη, contribute to suppression of HCV infection by regulating the integrity of OCLN. The disruption of OCLN integrity increased HCV infection. We also found that HCV disrupts ANX5-mediated OCLN integrity through downregulation of PKCα and PKCη expression, thereby promoting viral infection. We propose that HCV disrupts ANX5-mediated OCLN integrity to establish a persistent infection. The disruption of tight-junction assembly may play important roles in the progression of HCV-related liver diseases.

Nidogen-2 is a Novel Endogenous Ligand of LGR4 to Inhibit Vascular Calcification.

BACKGROUND: Vascular calcification is closely related to the all-cause mortality of cardiovascular events. Basement membrane protein nidogen-2 is a key component of the vascular extracellular matrix microenvironment and we recently found it is pivotal for the maintenance of contractile phenotype in vascular smooth muscle cells (VSMCs). However, whether nidogen-2 is involved in VSMCs osteochondrogenic transition and vascular calcification remains unclear. METHODS: VSMCs was treated with high-phosphate to study VSMC calcification in vitro. Three different mice models (5/6 nephrectomy-induced chronic renal failure, cholecalciferol-overload, and periadventitially administered with CaCl2) were used to study vascular calcification in vivo. Membrane protein interactome, coimmunoprecipitation, flow cytometric binding assay, surface plasmon resonance, G protein signaling, VSMCs calcium assays were performed to clarify the phenotype and elucidate the molecular mechanisms. RESULTS: Nidogen-2 protein levels were significantly reduced in calcified VSMCs and aortas from mice in different vascular calcification model. Nidogen-2 deficiency exacerbated high-phosphate-induced VSMC calcification, whereas the addition of purified nidogen-2 protein markedly alleviated VSMC calcification in vitro. Nidogen-2-/- mice exhibited aggravated aorta calcification compared to wild-type (WT) mice in response to 5/6 nephrectomy, cholecalciferol-overload, and CaCl2 administration. Further unbiased coimmunoprecipitation and interactome analysis of purified nidogen-2 and membrane protein in VSMCs revealed that nidogen-2 directly binds to LGR4 (leucine-rich repeat G-protein-coupled receptor 4) with KD value 26.77 nM. LGR4 deficiency in VSMCs in vitro or in vivo abolished the protective effect of nidogen-2 on vascular calcification. Of interest, nidogen-2 biased activated LGR4-Gαq-PKCα (protein kinase Cα)-AMPKα1 (AMP-activated protein kinase α1) signaling to counteract VSMCs osteogenic transition and mineralization. CONCLUSIONS: Nidogen-2 is a novel endogenous ligand of LGR4 that biased activated Gαq- PKCα-AMPKα1 signaling and inhibited vascular calcification.

Pulsatillic acid as a potential inhibitor of protein kinase C-alpha in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a global health concern with a significant impact on morbidity and mortality. Small molecule inhibitors targeting genetic mutations like EGFR and ALK have shown promise in NSCLC treatment. This study focuses on Protein Kinase C-alpha (PKCα), implicated in NSCLC pathogenesis. Overexpression of PKCα correlates with advanced disease stages. Preclinical studies suggest its inhibition can suppress NSCLC cell growth. The research employs molecular docking to identify Pulsatillic acid (PA) as a potential PKCα inhibitor. ADMET predictions support PA's candidacy and PASS analysis and Swiss Target Prediction reveal its biological properties. Fluorescence-based binding assays demonstrate PA's inhibitory potency on PKCα, aligning with molecular docking findings. Cytotoxicity assays show PA's minimal impact on HEK-293 cell viability, with an IC50 of 21.03 µM in A549 cells. mRNA expression analysis in A549 cells indicates PA's potential inhibitory effect on PKCα. In conclusion, this study highlights that PA may emerge as a promising therapeutic candidate for NSCLC, emphasizing the need for further research, validation, and exploration of its translational potential. The study contributes valuable insights into NSCLC treatment strategies, emphasizing the significance of targeting PKCα.

Teleocidin B-4, a PKC Activator, Upregulates Hypoxia-Inducible Factor 1 (HIF-1) Activity by Promoting the Accumulation of HIF-1α Protein via the PKCα/mTORC Signaling Pathway.

Hypoxia-inducible factor 1 (HIF-1) signaling is upregulated in an oxygen-dependent manner under hypoxic conditions. Activation of HIF-1 signaling increases the expression of HIF-1 target genes involved in cell survival, proliferation, and angiogenesis. Therefore, compounds that activate HIF-1 signaling have therapeutic potential in ischemic diseases. Screening for compounds that activate HIF-1 activity identified a microbial metabolite, teleocidin B-4, a PKC activator. Other PKC activators, such as TPA and 10-Me-Aplog-1, also activated HIF-1 activity. PKC activators induced HIF-1α protein accumulation through PKCα/mTORC activation. These results suggest that PKC activators without tumor-promoting activity have potential as therapeutic agents via HIF-1 target gene activation.

Click chemistry-enabled CRISPR screening reveals GSK3 as a regulator of PLD signaling.

Enzymes that produce second messengers are highly regulated. Revealing the mechanisms underlying such regulation is critical to understanding both how cells achieve specific signaling outcomes and return to homeostasis following a particular stimulus. Pooled genome-wide CRISPR screens are powerful unbiased approaches to elucidate regulatory networks, their principal limitation being the choice of phenotype selection. Here, we merge advances in bioorthogonal fluorescent labeling and CRISPR screening technologies to discover regulators of phospholipase D (PLD) signaling, which generates the potent lipid second messenger phosphatidic acid. Our results reveal glycogen synthase kinase 3 as a positive regulator of protein kinase C and PLD signaling. More generally, this work demonstrates how bioorthogonal, activity-based fluorescent tagging can expand the power of CRISPR screening to uncover mechanisms regulating specific enzyme-driven signaling pathways in mammalian cells.

Phosphorylated CPI-17 and MLC2 as Biomarkers of Coronary Artery Spasm-Induced Sudden Cardiac Death.

Coronary artery spasm (CAS) plays an important role in the pathogeneses of various ischemic heart diseases and has gradually become a common cause of life-threatening arrhythmia. The specific molecular mechanism of CAS has not been fully elucidated, nor are there any specific diagnostic markers for the condition. Therefore, this study aimed to examine the specific molecular mechanism underlying CAS, and screen for potential diagnostic markers. To this end, we successfully constructed a rat CAS model and achieved in vitro culture of a human coronary-artery smooth-muscle cell (hCASMC) contraction model. Possible molecular mechanisms by which protein kinase C (PKC) regulated CAS through the C kinase-potentiated protein phosphatase 1 inhibitor of 17 kDa (CPI-17)/myosin II regulatory light chain (MLC2) pathway were studied in vivo and in vitro to screen for potential molecular markers of CAS. We performed hematoxylin and eosin staining, myocardial zymogram, and transmission electron microscopy to determine myocardial and coronary artery injury in CAS rats. Then, using immunohistochemical staining, immunofluorescence staining, and Western blotting, we further demonstrated a potential molecular mechanism by which PKC regulated CAS via the CPI-17/MLC2 pathway. The results showed that membrane translocation of PKCα occurred in the coronary arteries of CAS rats. CPI-17/MLC2 signaling was observably activated in coronary arteries undergoing CAS. In addition, in vitro treatment of hCASMCs with angiotensin II (Ang II) increased PKCα membrane translocation while consistently activating CPI-17/MLC2 signaling. Conversely, GF-109203X and calphostin C, specific inhibitors of PKC, inactivated CPI-17/MLC2 signaling. We also collected the coronary artery tissues from deceased subjects suspected to have died of CAS and measured their levels of phosphorylated CPI-17 (p-CPI-17) and MLC2 (p-MLC2). Immunohistochemical staining was positive for p-CPI-17 and p-MLC2 in the tissues of these subjects. These findings suggest that PKCα induced CAS through the CPI-17/MLC2 pathway; therefore, p-CPI-17 and p-MLC2 could be used as potential markers for CAS. Our data provide novel evidence that therapeutic strategies against PKC or CPI-17/MLC2 signaling might be promising in the treatment of CAS.

The Ethyl Acetate Extract of Phyllanthus emblica L. Alleviates Diabetic Nephropathy in a Murine Model of Diabetes.

Oil-Gan is the fruit of the genus Phyllanthus emblica L. The fruits have excellent effects on health care and development values. There are many methods for the management of diabetic nephropathy (DN). However, there is a lack of effective drugs for treating DN throughout the disease course. The primary aim of this study was to examine the protective effects (including analyses of urine and blood, and inflammatory cytokine levels) and mechanisms of the ethyl acetate extract of P. emblica (EPE) on db/db mice, an animal model of diabetic nephropathy; the secondary aim was to examine the expression levels of p- protein kinase Cα (PKCα)/t-PKCα in the kidney and its downregulation of vascular endothelial growth factor (VEGF) and fibrosis gene transforming growth factor-ß1 (TGF-ß1) by Western blot analyses. Eight db/m mice were used as the control group. Forty db/db mice were randomly divided into five groups. Treatments included a vehicle, EPE1, EPE2, EPE3 (at doses of 100, 200, or 400 mg/kg EPE), or the comparative drug aminoguanidine for 8 weeks. After 8 weeks of treatment, the administration of EPE to db/db mice effectively controlled hyperglycemia and hyperinsulinemia by markedly lowering blood glucose, insulin, and glycosylated HbA1c levels. The administration of EPE to db/db mice decreased the levels of BUN and creatinine both in blood and urine and reduced urinary albumin excretion and the albumin creatine ratio (UACR) in urine. Moreover, EPE treatment decreased the blood levels of inflammatory cytokines, including kidney injury molecule-1 (KIM-1), C-reactive protein (CRP), and NLR family pyrin domain containing 3 (NLRP3). Our findings showed that EPE not only had antihyperglycemic effects but also improved renal function in db/db mice. A histological examination of the kidney by immunohistochemistry indicated that EPE can improve kidney function by ameliorating glomerular morphological damage following glomerular injury; alleviating proteinuria by upregulating the expression of nephrin, a biomarker of early glomerular damage; and inhibiting glomerular expansion and tubular fibrosis. Moreover, the administration of EPE to db/db mice increased the expression levels of p- PKCα/t-PKCα but decreased the expression levels of VEGF and renal fibrosis biomarkers (TGF-ß1, collagen IV, p-Smad2, p-Smad3, and Smad4), as shown by Western blot analyses. These results implied that EPE as a supplement has a protective effect against renal dysfunction through the amelioration of insulin resistance as well as the suppression of nephritis and fibrosis in a DN model.

A novel antiproliferative PKCα-Ras-ERK signaling axis in intestinal epithelial cells.

We have previously shown that the serine/threonine kinase PKCα triggers MAPK/ERK kinase (MEK)-dependent G1→S cell cycle arrest in intestinal epithelial cells, characterized by downregulation of cyclin D1 and inhibitor of DNA-binding protein 1 (Id1) and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Here, we use pharmacological inhibitors, genetic approaches, siRNA-mediated knockdown, and immunoprecipitation to further characterize antiproliferative ERK signaling in intestinal cells. We show that PKCα signaling intersects the Ras-Raf-MEK-ERK kinase cascade at the level of Ras small GTPases and that antiproliferative effects of PKCα require active Ras, Raf, MEK, and ERK, core ERK pathway components that are also essential for pro-proliferative ERK signaling induced by epidermal growth factor (EGF). However, PKCα-induced antiproliferative signaling differs from EGF signaling in that it is independent of the Ras guanine nucleotide exchange factors (Ras-GEFs), SOS1/2, and involves prolonged rather than transient ERK activation. PKCα forms complexes with A-Raf, B-Raf, and C-Raf that dissociate upon pathway activation, and all three Raf isoforms can mediate PKCα-induced antiproliferative effects. At least two PKCα-ERK pathways that collaborate to promote growth arrest were identified: one pathway requiring the Ras-GEF, RasGRP3, and H-Ras, leads to p21Cip1 upregulation, while additional pathway(s) mediate PKCα-induced cyclin D1 and Id1 downregulation. PKCα also induces ERK-dependent SOS1 phosphorylation, indicating possible negative crosstalk between antiproliferative and growth-promoting ERK signaling. Importantly, the spatiotemporal activation of PKCα and ERK in the intestinal epithelium in vivo supports the physiological relevance of these pathways and highlights the importance of antiproliferative ERK signaling to tissue homeostasis in the intestine.

O-GlcNAcylation stabilizes the autophagy-initiating kinase ULK1 by inhibiting chaperone-mediated autophagy upon HPV infection.

Human papillomaviruses (HPVs) cause a subset of head and neck squamous cell carcinomas (HNSCCs). Previously, we demonstrated that HPV16 oncogene E6 or E6/E7 transduction increases the abundance of O-linked ß-N-acetylglucosamine (O-GlcNAc) transferase (OGT), but OGT substrates affected by this increase are unclear. Here, we focus on the effects of O-GlcNAcylation on HPV-positive HNSCCs. We found that upon HPV infection, Unc-51-like kinase 1 (ULK1), an autophagy-initiating kinase, is hyper-O-GlcNAcylated, stabilized, and linked with autophagy elevation. Through mass spectrometry, we identified that ULK1 is O-GlcNAcylated at Ser409, which is distinct from the previously reported Thr635/Thr754 sites. It has been demonstrated that PKCα mediates phosphorylation of ULK1 at Ser423, which attenuates its stability by shunting ULK1 to the chaperone-mediated autophagy (CMA) pathway. Using biochemical assays, we demonstrate that ULK1 Ser409Ser410 O-GlcNAcylation antagonizes its phosphorylation at Ser423. Moreover, mutations of Ser409A and its neighboring site Ser410A (2A) render ULK1 less stable by promoting interaction with the CMA chaperone HSC70 (heat shock cognate 70 kDa protein). Furthermore, ULK1-2A mutants attenuate the association of ULK1 with STX17, which is vital for the fusion between autophagosomes and lysosomes. Analysis of The Cancer Genome Atlas (TCGA) database reveals that ULK1 is upregulated in HPV-positive HNSCCs, and its level positively correlates with HNSCC patient survival. Overall, our work demonstrates that O-GlcNAcylation of ULK1 is altered in response to environmental changes. O-GlcNAcylation of ULK1 at Ser409 and perhaps Ser410 stabilizes ULK1, which might underlie the molecular mechanism of HPV-positive HNSCC patient survival.

Novel Smooth Muscle Ca2+-Signaling Nanodomains in Blood Pressure Regulation.

BACKGROUND: Ca2+ signals in smooth muscle cells (SMCs) contribute to vascular resistance and control blood pressure. Increased vascular resistance in hypertension has been attributed to impaired SMC Ca2+ signaling mechanisms. In this regard, transient receptor potential vanilloid 4 (TRPV4SMC) ion channels are a crucial Ca2+ entry pathway in SMCs. However, their role in blood pressure regulation has not been identified. METHODS: We used SMC-specific TRPV4-/- (TRPV4SMC-/-) mice to assess the role of TRPV4SMC channels in blood pressure regulation. We determined the contribution of TRPV4SMC channels to the constrictor effect of α1 adrenergic receptor (α1AR) stimulation and elevated intraluminal pressure: 2 main physiologic stimuli that constrict resistance-sized arteries. The contribution of spatially separated TRPV4SMC channel subpopulations to elevated blood pressure in hypertension was evaluated in angiotensin II-infused mice and patients with hypertension. RESULTS: We provide first evidence that TRPV4SMC channel activity elevates resting blood pressure in normal mice. α1AR stimulation activated TRPV4SMC channels through PKCα (protein kinase Cα) signaling, which contributed significantly to vasoconstriction and blood pressure elevation. Intraluminal pressure-induced TRPV4SMC channel activity opposed vasoconstriction through activation of Ca2+-sensitive K+ (BK) channels, indicating functionally opposite pools of TRPV4SMC channels. Superresolution imaging of SMCs revealed spatially separated α1AR:TRPV4 and TRPV4:BK nanodomains in SMCs. These data suggest that spatially separated α1AR-TRPV4SMC and intraluminal pressure-TRPV4SMC-BK channel signaling have opposite effects on blood pressure, with α1AR-TRPV4SMC signaling dominating under resting conditions. Furthermore, in patients with hypertension and a mouse model of hypertension, constrictor α1AR-PKCα-TRPV4 signaling was upregulated, whereas dilator pressure-TRPV4-BK channel signaling was disrupted, thereby increasing vasoconstriction and elevating blood pressure. CONCLUSIONS: Our data identify novel smooth muscle Ca2+-signaling nanodomains that regulate blood pressure and demonstrate their impairment in hypertension.

Inhibition of protein kinase C-α and activation of ezrin by Lactobacillus acidophilus restore Na+/H+ exchange activity and fluid absorption in db/db mice.

Gastrointestinal (GI) complications, including diarrhea, constipation, and gastroparesis, are common in patients with diabetes. Dysregulation of the Na+/H+ exchanger NHE3 in the intestine is linked to diarrhea and constipation, and recent studies showed that NHE3 expression is reduced in type 1 diabetes and metformin causes diarrhea in the db/db mouse model of type 2 diabetes (T2D) via inhibition of NHE3. In this study, we investigated whether NHE3 expression is altered in type 2 diabetic intestine and the underlying mechanism that dysregulates NHE3. NHE3 expression in the brush border membrane (BBM) of the intestine of diabetic mice and humans was decreased. Protein kinase C (PKC) activation is associated with pathologies of diabetes, and immunofluorescence (IF) analysis revealed increased BBM PKCα abundance. Inhibition of PKCα increased NHE3 BBM abundance and NHE3-mediated intestinal fluid absorption in db/db mice. Previous studies have shown that Lactobacillus acidophilus (LA) stimulates intestinal ion transporters. LA increased NHE3 BBM expression and mitigated metformin-mediated inhibition of NHE3 in vitro and in vivo. To understand the underlying mechanism of LA-mediated stimulation of NHE3, we used Caco-2bbe cells overexpressing PKCα that mimic the elevated state of PKCα in T2D. LA diminished PKCα BBM expression, increased phosphorylation of ezrin, and the interaction of NHE3 with NHE regulatory factor 2 (NHERF2). In addition, inhibition of PKCι blocked phosphorylation of ezrin and activation of NHE3 by LA. These findings demonstrate that NHE3 is downregulated in T2D, and LA restores NHE3 expression via regulation of PKCα, PKCι, and ezrin.NEW & NOTEWORTHY We used mouse models of type 2 diabetes (T2D) and human patient-derived samples to show that Na+/H+ exchanger 3 (NHE3) expression is decreased in T2D. We show that protein kinase C-α (PKCα) is activated in diabetes and inhibition of PKCα increased NHE3 expression and mitigates diarrhea. We show that Lactobacillus acidophilus (LA) stimulates NHE3 via inhibition of PKCα and phosphorylation of ezrin.