PKCα Expression Research Articles

PKCα regulates the secretion of PDL1-carrying small extracellular vesicles in a p53-dependent manner

Small extracellular vesicles (sEVs), carrying PD-L1, have been implicated in immune evasion and tumor progression. However, understanding how PD-L1 sEVs are secreted still needs to be improved. We found that the secretion dynamics of PD-L1 sEVs is similar to that of other sEVs. Intracellular calcium and the associated downstream PKC signaling plays pivotal roles in releasing PD-L1 sEVs in non-small cell lung cancer cells (NSCLC). Particularly, we observed that knocking down PKCα has profound impacts on PD-L1 sEVs secretion, especially in the resting state and in the activated state, when induced by an intracellular calcium rise. Furthermore, our study revealed that PKCα regulates PD-L1 expression and PD-L1 sEVs secretion by influencing STAT1 phosphorylation and nuclear translocation in a p53-dependent manner. Notably, p53 can regulate STAT1 phosphorylation and nuclear localization, but it does not affect PKCα expression. This suggests that PKCα plays a significant role in regulating PD-L1 expression. Our findings suggest that targeting PKCα to modulate PD-L1 dynamics in NSCLC may be a promising therapeutic strategy to enhance the efficacy of immunotherapeutic interventions.

PKCα Activation via the Thyroid Hormone Membrane Receptor Is Key to Thyroid Cancer Growth.

Thyroid carcinoma (TC) is the most common endocrine neoplasia, with its incidence increasing in the last 40 years worldwide. The determination of genetic and/or protein markers for thyroid carcinoma could increase diagnostic precision. Accumulated evidence shows that Protein kinase C alpha (PKCα) contributes to tumorigenesis and therapy resistance in cancer. However, the role of PKCα in TC remains poorly studied. Our group and others have demonstrated that PKCs can mediate the proliferative effects of thyroid hormones (THs) through their membrane receptor, the integrin αvβ3, in several cancer types. We found that PKCα is overexpressed in TC cell lines, and it also appeared as the predominant expressed isoform in public databases of TC patients. PKCα-depleted cells significantly reduced THs-induced proliferation, mediated by the integrin αvβ3 receptor, through AKT and Erk activation. In databases of TC patients, higher PKCα expression was associated with lower overall survival. Further analyses showed a positive correlation between PKCα and genes from the MAPK and PI3K-Akt pathways. Finally, immunohistochemical analysis showed abnormal upregulation of PKCα in human thyroid tumors. Our findings establish a potential role for PKCα in the control of hormone-induced proliferation that can be explored as a therapeutic and/or diagnostic target for TC.

Rotigaptide inhibits spontaneous contractions of gastric smooth muscle in diabetic rats via the PKCα-Cx43 pathway.

The study aimed to investigate the effect of rotigaptide (ZP123) on spontaneous contractions of gastric smooth muscle in diabetic rats and explore the underlying mechanisms. Twelve rats were randomly divided into model and normal control groups. Changes in gastric smooth muscle spontaneous contractions in each group were observed. Western blot analysis was performed to detect Cx43 and PKCα expression. Rat gastric smooth muscle cells were cultured in vitro and divided into normal glucose, high glucose and high glucose+rotigaptide group. The intracellular Ca2+ content was observed by immunofluorescence. The amplitude and frequency of gastric smooth muscle spontaneous contractions were reduced in the model group than the normal control group (all p < .01), which were reduced after rotigatide treatment than before treatment in the model group (all p < .01). The model+rotigaptide group showed decreased membrane expression of Cx43, increased cytoplasmic expression of Cx43, increased membrane expression of p-PKCα Thr497 and lower membrane/cytoplasm ratio of Cx43 expression compared with the model group (all p < .01). The intracellular Ca2+ content was increased in the high glucose group than the normal glucose group (p < .01), while no significant difference was observed between the high glucose+rotigaptide and high glucose groups. Our findings suggest that rotigatide can stabilize the intracellular Ca2+ concentration in gastric smooth muscle cells under high glucose condition by upregulating PKCα activity and downregulating the number of GJs and the opening rate of GJ hemichannels through the PKCα-Cx43 pathway, thus inhibiting spontaneous contractions of gastric smooth muscle in diabetic rats.

Experimental study on treatment of retinitis pigmentosa by inducing Müller cell reprogramming with Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma

This study aims to explore the effect of Lycii Fructus and Salviae Miltiorrhizae Radix et Rhizoma(LFSMR), a drug pair possesses the function of nourishing Yin, promoting blood circulation, and brightening the eyes, in treating retinitis pigmentosa(RP)by inhibiting the gliosis of Müller cells(MCs) and inducing their reprogramming and differentiation into various types of retinal nerve cells. Twelve C57 mice were used as the normal control group, and 48 transgenic RP(rd10) mice were randomly divided into the model group, positive control group, and low and high dose LFSMR groups, with 12 mice in each group. HE staining was used to detect pathological changes in the retina, and an electroretinogram was used to detect retinal function. Retinal optical coherence tomography was used to detect retinal thickness and perform fundus photography, and laser speckle perfusion imaging was used to detect local retinal blood flow. Digital PCR was used to detect gene expression related to retinal nerve cells, and immunofluorescence was used to detect protein expression related to retinal nerve cells. LFSMR could significantly improve the pathological changes, increase the amplitude of a and b waves, increase the retinal thickness, restore retinal damage, and increase retinal blood flow in mice with RP lesions. LFSMR could also significantly inhibit the m RNA expression of the glial fibrillary acidic protein( GFAP) during the pathogenesis of RP and upregulate m RNA expression of sex determining region Y box protein 2(SOX2), paired box protein 6(Pax6),rhodopsin, protein kinase C-α(PKCα), syntaxin, and thymic cell antigen 1. 1(Thy1. 1). LFSMR could significantly inhibit GFAP protein expression and enhance protein expression of SOX2, Pax6, rhodopsin, PKCα, syntaxin, and Thy1. 1. It could also reverse the pathological changes in the retina of rd10 mice, improve retinal function and fundus performance, increase retinal thickness, enhance local retinal blood flow, and exert therapeutic effects on RP. The mechanism of action of LFSMR may be related to inhibiting the gliosis of MCs and promoting their reprogramming and differentiation into various types of retinal nerve cells.

α-Hederin induces paraptosis by targeting GPCRs to activate Ca2+/MAPK signaling pathway in colorectal cancer.

Non-apoptotic cell death is presently emerging as a potential direction to overcome the apoptosis resistance of cancer cells. In the current study, a natural plant agent α-hederin (α-hed) induces caspase-independent paraptotic modes of cell death. The present study is aimed to investigate the role of α-hed induces paraptosis and the associated mechanism of it. The cell proliferation was detected by CCK-8. The cytoplasm organelles were observed under electron microscope. Calcium (Ca2+) level was detected by flow cytometry. Swiss Target Prediction tool analyzed the potential molecule targets of α-hed. Molecular docking methods were used to evaluate binding abilities of α-hed with targets. The expressions of genes and proteins were analyzed by RT-qPCR, western blotting, immunofluorescence, and immunohistochemistry. Xenograft models in nude mice were established to evaluate the anticancer effects invivo. α-hed exerted significant cytotoxicity against a panel of CRC cell lines by inhibiting proliferation. Besides, it induced cytoplasmic vacuolation in all CRC cells. Electron microscopy images showed the aberrant dilation of endoplasmic reticulum and mitochondria. Both mRNA and protein expressions of Alg-2 interacting proteinX (Alix), the marker of paraptosis, were inhibited by α-hed. Besides, both Swiss prediction and molecular docking showed that the structure of α-hed could tightly target to GPCRs. GPCRs were reported to activate the phospholipase C (PLC)-β3/ inositol 1,4,5-trisphosphate receptor (IP3R)/ Ca2+/ protein kinase C alpha (PKCα) pathway, and we then found all proteins and mRNA expressions of PLCβ3, IP3R, and PKCα were increased by α-hed. After blocking the GPCR signaling, α-hed could not elevate Ca2+ level and showed less CRC cell cytotoxicity. MAPK cascade is the symbol of paraptosis, and we then demonstrated that α-hed activated MAPK cascade by elevating Ca2+ flux. Since non-apoptotic cell death is presently emerging as a potential direction to overcome chemo-drug resistance, we then found α-hed also induced paraptosis in 5-fluorouracil-resistant (5-FU-R) CRC cells, and it reduced the growth of 5-FU-R CRC xenografts. Collectively, our findings proved α-hed as a promising candidate for inducing non-apoptotic cell death, paraptosis. It may overcome the resistance of apoptotic-based chemo-resistance in CRC.

Transcending Traditional Treatment: The Therapeutical Potential of Nanovesicles for Transdermal Baclofen Delivery in Repeated Traumatic Brain Injury.

The repositioning of previously approved drugs is occupying the researchers' plans. Baclofen (Bac) was our candidate for its established neuroprotective capacity, with a proposal of efficient drug delivery as non-ionic surfactant-based nanovesicles (NISNV) formulae against mild repetitive traumatic brain injury (mRTBI) in rats, thus reducing the number of orally or injected medications, especially in severely comatose patients or pediatrics. A (23) factorial design was implemented for confining Bac-loaded NISNV formulae, where a bunch of variables were inspected. An in-vivo experiment was done to test the prepared formula's efficacy transdermally. The following parameters were measured: brain expression of gamma amino butyric acid B (GABAB), protein kinase C- α (PKC-α), focal adhesion kinase (FAK), TNF-α and nuclear factor kappa B (NF-κB) p65, malondialdehyde (MDA), superoxide dismutase (SOD), and histopathology. The particle size (PS) and entrapment efficiency percent (EE%) speckled from 60.40±0.28% to 88.02±0.01% for the former and 174.64±0.93 to 1174.50±3.54 nm for the latter. In vitro release% after 8 hours ranged from 63.25±5.47% to 84.79±3.75%. The optimized formula (F4) illustrated desirability=1, with 630.09±3.53 µg/cm2 of Bac permeated over 8 hours, which equates to 100% of Bac. Bac post-trauma treatment restored brain expression of GABAB and PKC-α, while decreasing FAK. Besides enhancing the histological findings, the anti-inflammatory effect was clear by decreasing TNF-α and NF-κB p65. Consequently, significant antioxidant sequelae were revealed herein by diminishing MDA levels and restoring SOD activity. Transdermal delivery of Bac-loaded niosomes confirmed neuroprotection and succeeded in surpassing skin-to-brain barriers, which makes it a promising therapeutic option for repeated traumas.

Glycyrrhizic acid rebalances mitochondrial dynamics to mitigate hepatotoxicity induced by triptolide

Triptolide (TP) has various pharmacological activities, but its hepatotoxicity limits its therapeutic potential. Licorice has been evaluated to harmonize various medicines, and its active ingredient glycyrrhizic acid (GA) has been identified for its detoxification effect. In this study, we demonstrated that the combination of GA and TP (G&T) could mitigate TP-induced hepatotoxicity. To explore TP toxicity and G&T detoxification mechanisms, transcriptomics and proteomics have been utilized. TP was found to promote the expression of PKCα and FIS1, accompanied by an increase in mitochondrial fission. After pretreatment with GA, improvements were observed in TP-induced mitochondrial fission, as well as the expression of active-caspase3, and cytochrome c release. Silencing PKCα and FIS1 has a similar effect to GA on the regulation of mitochondria-dependent apoptosis and mitochondrial fission. In conclusion, PKCα and FIS1-involved mitochondrial dynamic regulation is associated with TP-induced hepatotoxicity. The G&T combination may be an effective strategy for ameliorating TP-induced toxicity.

Proteomics-based screening of AKR1B1 as a therapeutic target and validation study for sepsis-associated acute kidney injury.

Sepsis and sepsis-associated acute kidney injury (SA-AKI) pose significant global health challenges, necessitating the development of innovative therapeutic strategies. Dysregulated protein expression has been implicated in the initiation and progression of sepsis and SA-AKI. Identifying potential protein targets and modulating their expression is crucial for exploring alternative therapies. We established an SA-AKI rat model using cecum ligation perforation (CLP) and employed differential proteomic techniques to identify protein expression variations in kidney tissues. Aldose reductase (AKR1B1) emerged as a promising target. The SA-AKI rat model received treatment with the aldose reductase inhibitor (ARI), epalrestat. Blood urea nitrogen (BUN) and creatinine (CRE) levels, as well as IL-1β, IL-6 and TNF-α levels in the serum and kidney tissues, were monitored. Hematoxylin-eosin (H-E) staining and a pathological damage scoring scale assessed renal tissue damage, while protein blotting determined PKC (protein kinase C)/NF-κB pathway protein expression. Differential proteomics revealed significant downregulation of seven proteins and upregulation of 17 proteins in the SA-AKI rat model renal tissues. AKR1B1 protein expression was notably elevated, confirmed by Western blot. ARI prophylactic administration and ARI treatment groups exhibited reduced renal injury, low BUN and CRE levels and decreased IL-1β, IL-6 and TNF-α levels compared to the CLP group. These changes were statistically significant (P<0.05). AKR1B1, PKC-α, and NF-κB protein expression levels were also lowered in the ARI prophylactic administration and ARI treatment groups compared to the CLP group (P<0.05). Epalrestat appeared to inhibit the PKC/NF-κB inflammatory pathway by inhibiting AKR1B1, resulting in reduced inflammatory cytokine levels in renal tissues and blood. This mitigated renal tissue injuries and improved the systemic inflammatory response in the severe sepsis rat model. Consequently, AKR1B1 holds promise as a target for treating sepsis-associated acute kidney injuries.

Puerarin alleviates the high glucose-induced oxidative stress via the RAGE/PKC/NOX4 axis in renal mesangial cells.

Diabetic nephropathy (DN) is a severe microvascular complication of diabetes, of which progression is related to high glucose (HG)-induced oxidative stress in renal mesangial cells. Our study aims to explore the antioxidant activity and the underlying mechanism of Puerarin (Pu) in renal mesangial cells exposed to HG. After the cells finished different treatments, DCFH-DA was used to detect the generation of ROS while the expression of AGE, MDA, SOD, and GSH-PX was measured by the ELISA and corresponding kits. The cell morphology was captured by optical microscopy. The mRNA expressions of RAGE, PKCα, PKCβ, PKCγ, and NOX4 were calculated by RT-PCR assays, while the protein expressions of RAGE, NOX4, and PKCβ were quantified via western blotting. Compared with the normal glucose (NG) group, the ROS level, SOD activity, and GSH-PX expression were markedly reduced in the HG group while the MDA expression was increased in the HG group. Then, Pu treatment was proved to significantly prevent the HG-induced up-regulation of ROS level, MDA expression, and down-regulation of SOD activity and GSH-PX expression. Besides, Pu treatment can notably inhibit the AGE expression and reverse the increased RAGE, PKCβ, and NOX4 expressions by HG environment at both RNA and protein levels. Moreover, the antioxidant effect of Pu against access glucose could not be observed in PKCβ knockdown cells. Pu can alleviate the HG-induced oxidative stress via the RAGE/PKC/NOX4 axis in renal mesangial cells, which innovatively suggests the therapeutic potential of Pu for DN treatment.

Sphincter of Oddi Dysfunction Induces Gallstone by Inhibiting the Expression of ABCB11 via PKC-α.

The abnormal increase of Oddi sphincter pressure and total bile duct pressure may play an important role in the formation of cholesterol stones, but the specific molecular mechanism is still unclear. This study aims to investigate it through in vitro and in vivo experiments. A mouse model of Oddi sphincter dysfunction was constructed by stone-inducing diet. We compared the two groups with PKC-α inhibitor GÖ6976 and PKC-α agonist thymeleatoxin. Oddi sphincter pressure and total bile duct pressure were measured. Biochemical analysis of total cholesterol, bile acid and bilirubin was then conducted. The histopathologic changes of bile duct were observed by HE staining and the ultrastructure of liver cells and surrounding tissues was observed by transmission electron microscopy. Through the above experiments, we found that the change of PKC-α expression may affect the formation process of gallstones. The relationship between PKC-α and ABCB11 was further verified by in vitro and in vivo experiments. Our results suggest that ABCB11 and PKC-α are co-expressed in the tubule membrane of hepatocytes and interact with each other in hepatocytes. The high cholesterol diet further enhances the activation of PKC-α and thus reduces the expression of ABCB11. The formation of cholesterol stones is associated with the down-regulation of ABCB11 expression in the tubule membrane of hepatocytes due to kinase signaling. This is the first study to demonstrate that sphincter of Oddi dysfunction induces gallstones through PKC-α inhibition of ABCB11 expression.

Adipose transplantation improves olfactory function and neurogenesis via PKCα-involved lipid metabolism in Seipin Knockout mice

BackgroundLipodystrophy-associated metabolic disorders caused by Seipin deficiency lead to not only severe lipodystrophy but also neurological disorders. However, the underlying mechanism of Seipin deficiency-induced neuropathy is not well elucidated, and the possible restorative strategy needs to be explored.MethodsIn the present study, we used Seipin knockout (KO) mice, combined with transcriptome analysis, mass spectrometry imaging, neurobehavior test, and cellular and molecular assay to investigate the systemic lipid metabolic abnormalities in lipodystrophic mice model and their effects on adult neurogenesis in the subventricular zone (SVZ) and olfactory function. After subcutaneous adipose tissue (AT) transplantation, metabolic and neurological function was measured in Seipin KO mice to clarify whether restoring lipid metabolic homeostasis would improve neurobehavior.ResultsIt was found that Seipin KO mice presented the ectopic accumulation of lipids in the lateral ventricle, accompanied by decreased neurogenesis in adult SVZ, diminished new neuron formation in the olfactory bulb, and impaired olfactory-related memory. Transcriptome analysis showed that the differentially expressed genes (DEGs) in SVZ of adult Seipin KO mice were significantly enriched in lipid metabolism. Mass spectrometry imaging showed that the levels of glycerophospholipid and diglyceride (DG) were significantly increased. Furthermore, we found that AT transplantation rescued the abnormality of peripheral metabolism in Seipin KO mice and ameliorated the ectopic lipid accumulation, concomitant with restoration of the SVZ neurogenesis and olfactory function. Mechanistically, PKCα expression was up-regulated in SVZ tissues of Seipin KO mice, which may be a potential mediator between lipid dysregulation and neurological disorder. DG analogue (Dic8) can up-regulate PKCα and inhibit the proliferation and differentiation of neural stem cells (NSCs) in vitro, while PKCα inhibitor can block this effect.ConclusionThis study demonstrates that Seipin deficiency can lead to systemic lipid disorder with concomitant SVZ neurogenesis and impaired olfactory memory. However, AT restores lipid homeostasis and neurogenesis. PKCα is a key mediator mediating Seipin KO-induced abnormal lipid metabolism and impaired neurogenesis in the SVZ, and inhibition of PKCα can restore the impaired neurogenesis. This work reveals the underlying mechanism of Seipin deficiency-induced neurological dysfunction and provides new ideas for the treatment of neurological dysfunction caused by metabolic disorders.

PKCα Isoform Inhibits Insulin Signaling and Aggravates Neuronal Insulin Resistance.

Overexpression of PKCα has been linked to inhibit insulin signaling disrupting IRS-1 and Akt phosphorylations in skeletal muscle. PKCα inhibits IRS-1 and Akt phosphorylations, but not required for insulin-stimulated glucose transport in skeletal muscles. Inhibition of PKCα increased whereas in some studies decreased GLUT-4 levels at the plasma membrane in skeletal muscles and adipocytes. Controversial studies have reported opposite expression pattern of PKCα expression in insulin-resistant skeletal muscles. These findings indicate that the role of PKCα on insulin signaling is controversial and could be tissue specific. Evidently, studies are required to decipher the role of PKCα in regulating insulin signaling and preferably in other cellular systems. Utilizing neuronal cells, like Neuro-2a, SHSY-5Y and insulin-resistant diabetic mice brain tissues; we have demonstrated that PKCα inhibits insulin signaling, through IRS-Akt pathway in PP2A-dependent mechanism by an AS160-independent route involving 14-3-3ζ. Inhibition and silencing of PKCα improves insulin sensitivity by increasing GLUT-4 translocation to the plasma membrane and glucose uptake. PKCα regulates GSK3 isoforms in an opposite manner in insulin-sensitive and in insulin-resistant condition. Higher activity of PKCα aggravates insulin-resistant neuronal diabetic condition through GSK3β but not GSK3α. Our results mechanistically explored the contribution of PKCα in regulating neuronal insulin resistance and diabetes, which opens up new avenues in dealing with metabolic disorders and neurodegenerative disorders.

Sperm immotility is associated with epididymis metabolism disorder in mice under obstructive azoospermia.

Obstructive azoospermia (OA) accounts for approximately 40% of males who suffer from azoospermia of male infertility. Currently, available treatment for OA consists of reproductive tract surgical reconstruction and sperm retrieval from the testis. However, both treatments result in low fertility compared to normal pregnancy, and the main reason remains largely unknown. Previous studies have shown that the quality of sperm retrieved from OA patients is poor compared with normal adult males but without an in-depth study. Herein, we generated a mouse OA model with vasectomy to evaluate sperm quality systematically. Our results showed that the testis had normal spermatogenesis but increased apoptotic activity in both OA patients and mice. More importantly, epididymal morphology was abnormal, with swollen epididymal tubules and vacuole-like principal cells. Especially, sperm retrieved from the epididymis of OA mice showed poor motility and low fertilization ability in vitro. Using mass spectrometry in epididymal fluid, we found differences in the expression of key proteins for sperm maturation, such as Angiotensinogen (AGT), rhophilin-associated tail protein 1 (ROPN1), NPC intracellular cholesterol transporter 2 (NPC2), and prominin 1 (PROM1). Furthermore, our results demonstrated that AGT, secreted by epididymal principal cells, could regulate sperm motility by managing PKCα expression to modify sperm phosphorylation. In conclusion, our data evaluate sperm quality systematically in OA mice and contribute to the understanding between the sperm and epididymis, which may provide novel insight into treating male infertility.

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.

PKC-βII is downregulated in the premature ovarian failure SD rat model.

We investigated the role of protein kinase c (PKC) -α and -β during the ovarian follicular dynamics using estrous cycle, gonadotropin-induced ovulation, and antral follicle culture, 4-vinylcyclohexene diepoxide (VCD)-induced premature ovarian failure (POF) in the SD rat models. We found the higher activity of PKC during the proestrus stage along with expression of PKC-α during the estrus and metestrus stages of the estrous cycle while PKC-β expression was increased during the diestrus, proestrus, and estrus stages. In response to pregnant mare gonadotropin (PMSG)-induced follicular recruitment and ovulation, the phosphorylated (Thr-642) PKC-β was increased. PKC activity inhibition by hispidin during the proestrus stage resulted in decreased antral follicles and corpus luteum. Treatment with hispidin resulted in the downregulation of granulosa cell (GC) biomarker, follicle stimulating hormone receptor (FSHR) expression in the cultured pre-antral follicle.During the forskolin-induced luteinization of human granulosa cells, the expression level of PKC-α and β (I and II) was decreased. In the POF condition, the activity of total PKC and the expression levels of PKC-α and β (I and II) were increased. Immunostaining depicted ubiquitous expression of PKC-α in the ovary during the estrous cycle and POF conditions. Taken together, we conclude the association of PKC-α and -β (I and II) during ovarian follicular dynamics where the expression level of PKC-α is increased, but the expression level of PKC-β (I and II) is suppressed in the POF condition in the SD rat model.

Nutritional Modulation of Guinea Pig Skin Hyperproliferation by Essential Fatty Acid Deficiency is Associated with Selective Down Regulation of Protein Kinase C-β

In a previous study we demonstrated that 13-hydroxyoctadecadienoic acid (13-HODE), a 15-lipoxygenase metabolite of linoleic acid is incorporated into epidermal phosphatidyl 4,5-bisphosphate (PtdIns 4,5-P2) and released as 13-HODE-containing-diacylglycerol (13-HODE-DAG). In vitro, 13-HODE-DAG was shown to selectively inhibit epidermal total protein kinase C (PKC-β) activity. To determine whether these observations are relevant in vivo, guinea pigs were made essential fatty acid deficient (EFAD) by feeding them a basal diet supplemented with 4% hydrogenated coconut oil for 8 wk. Tissue levels of putative 13-HODE-DAG, protein kinase C (PKC) isozymes and tissue hyperproliferation were determined in the epidermal preparations from skin of control safflower oil-fed guinea pigs, those fed EFAD diet and those fed EFAD diet followed by the control diet for 2 wk. Our data revealed that cutaneous 13-HODE and 13-HODE-DAG were significantly lower in EFAD animals than in safflower-fed controls. These reductions were associated with both elevated epidermal hyperproliferation and elevated expressions and activities of PKC-α and β-isozymes. Refeeding the animals with safflower oil for 2 wk replenished tissue levels of 13-HODE-DAG, which inversely correlated with the selective down regulation of PKC-β expression and activity and the reversal of hyperproliferation. In contrast, although the expression and activity of PKC-α was elevated in the epidermis of the EFAD guinea pigs, this elevated PKC-α expression was not down regulated after refeeding the safflower oil diet to the animals. These results suggest that the epidermal level of 13-HODE plays a role in vivo in modulating cutaneous hyperproliferation via the generation of 13-HODE-DAG via signal transduction and its selective suppression of PKC-β isozyme.

Crosstalk between protein kinase C α and transforming growth factor β 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 β (TGFβ) are negative regulators of proliferation with tumor suppressor properties in the intestine. Here, we identify novel crosstalk between PKCα and TGFβ signaling. RNA-Seq analysis of nontransformed intestinal crypt-like cells and colorectal cancer cells identified TGFβ receptor 1 (TGFβR1) as a target of PKCα signaling. RT-PCR and immunoblot analysis confirmed that PKCα positively regulates TGFβR1 mRNA and protein expression in these cells. Effects on TGFβR1 were dependent on Ras-extracellular signal-regulated kinase 1/2 (ERK) signaling. Nascent RNA and promoter-reporter analysis indicated that PKCα induces TGFβR1 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 TGFβR1 gene and induction of Runx2 expression. Thus, we have identified a novel PKCα→ERK→Runx2→TGFβR1 signaling axis. In further support of a link between PKCα and TGFβ signaling, PKCα knockdown reduced the ability of TGFβ to induce SMAD2 phosphorylation and cell cycle arrest, and inhibition of TGFβR1 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 TGFβR1 mRNA expression in human colorectal cancer. PKCα also regulated TGFβR1 in endometrial cancer cells, and PKCα, Runx2, and TGFβR1 expression correlates in uterine tumors, indicating that crosstalk between PKCα and TGFβ signaling may be a common mechanism in diverse epithelial tissues.

APOBEC3B Is Co-Expressed with PKCα/NF-κB in Oral and Oropharyngeal Squamous Cell Carcinomas.

The enzymatic activity of APOBEC3B (A3B) has been implicated as a prime source of mutagenesis in head and neck squamous cell carcinoma (HNSCC). The expression of Protein Kinase C α (PKCα) and Nuclear Factor-κΒ p65 (NF-κΒ p65) has been linked to the activation of the classical and the non-canonical NF-κB signaling pathways, respectively, both of which have been shown to lead to the upregulation of A3B. Accordingly, the aim of the present study was to evaluate the expression of PKCα, NF-κΒ p65 and A3B in non-HPV related oral and oropharyngeal squamous cell carcinomas (SCC), by means of immunohistochemistry and in silico methods. PKCα was expressed in 29/36 (80%) cases of oral and oropharyngeal SCCs, with 25 (69%) cases showing a PKCα+/A3B+ phenotype and only 6/36 (17%) cases showing a PKCα-/A3B+ phenotype. Εxpression of NF-κB p65 was seen in 33/35 (94%) cases of oral and oropharyngeal SCCs, with 30/35 (86%) cases showing an NF-κB p65+/A3B+ phenotype and only 2/35 (6%) cases showing an NF-κB p65-/A3B+ phenotype. In addition, mRNA expression analysis, using the UALCAN database, revealed strong expression of all three genes. These findings indicate that the expression of A3B is associated with PKCα/NF-κB p65 expression and suggest a potential role for the PKC/NF-κB signaling pathway in the development of oral and oropharyngeal cancer.

Increased Nuclear FOXP2 Is Related to Reduced Neural Stem Cell Number and Increased Neurogenesis in the Dorsal Telencephalon of Embryos of Diabetic Rats through Histamine H1 Receptors.

Diabetic rat embryos have increased cortical neurogenesis and neuron maturation, and their offspring presented altered neuron polarity, lamination, and diminished neuron excitability. The FOXP2 overexpression results in higher cortical neurogenesis by increasing the transition of radial glia to the intermediate progenitor. Similarly, histamine through H1-receptor activation increases cortical neuron differentiation. Indeed, blocking the H1-receptor by the systemic administration of chlorpheniramine to diabetic pregnant rats prevents increased neurogenesis. Here, we explore the relationship between the H1-receptor and FOXP2 on embryo neurogenesis from diabetic dams. Through qRT-PCR, Western blot, immunohistofluorescence, and flow cytometry, we showed an increased FOXP2 expression and nuclear localization, a reduced Nestin expression and -positive cells number, and a higher PKCα expression in the cortical neuroepithelium of fourteen-day-old embryos from diabetic rats. Interestingly, this scenario was prevented by the chlorpheniramine systemic administration to diabetic pregnant rats at embryo day twelve. These data, together with the bioinformatic analysis, suggest that higher H1-receptor activity in embryos under high glucose increases FOXP2 nuclear translocation, presumably through PKCα phosphorylation, impairing the transition of radial glia to intermediate progenitor and increasing neuron differentiation in embryos of diabetic rats.

The Effect of Long-Term Tamsulosin Monotherapy and Tamsulosin - Dutasteride Combination Therapy on PKC-α Enzyme Expression in Prostate Stromal Tissue.

The α-adrenergic receptor antagonist is the most effective medical therapy to reduce the dynamic component in patients with BPH. However, long-term administration of receptor antagonists can cause upregulation of mRNA receptor expression, resulting in tolerance of drug effectiveness. PKC-α is involved in the process of prostate smooth muscle contraction through activation of the voltage-gated Ca2+ conducted canal, influenced by androgen hormones, especially testosterone, and has an isoform with Twist1, a transcription factor that plays a role in up-regulation of androgen receptors. The aim of the study was to compare the effect of long-term tamsulosin monotherapy and tamsulosin - dutasteride combination therapy in PKC-α enzyme expression in prostate stromal tissue of Rattus norvegicus rats of Wistar strain. Out of 80 samples of Rattus norvegicus rats were divided into 8 groups with different interventions: negative control group, positive control group, tamsulosin monotherapy administration for 1 day, 3 day, and 6 day groups, and tamsulosin - dutasteride combination therapy for 1 day, 3 day, and 6 day groups. BPH was induced with 3 mg/kg of testosterone proprionate for 3 weeks, continued with drugs administration according to intervention grouping. Prostate stromal tissue was taken and prepared for PKC-α enzyme measurement with ELISA method. There was a significant difference (p<0.05) in the effect of tamsulosin monotherapy and tamsulosin-dutasteride combination therapy on the PKC-α expression. There was a strong positive relationship between the duration of tamsulosin-dutasteride combination therapy on the PKC-α expression, which means the longer the duration of the combination of tamsulosin-dutasteride combination the higher the PKC-α expression. Administration of long-term tamsulosin - dutasteride combination therapy causes upregulation PKC-α expression more than tamsulosin only.