Unveiling the Role of Protein Kinase C θ in Porcine Epidemic Diarrhea Virus Replication: Insights from Genome-Wide CRISPR/Cas9 Library Screening.

Porcine epidemic diarrhea virus (PEDV), a member of the Alpha-coronavirus genus in the Coronaviridae family, induces acute diarrhea, vomiting, and dehydration in neonatal piglets. This study aimed to investigate the genetic dependencies of PEDV and identify potential therapeutic targets by using a single-guide RNA (sgRNA) lentiviral library to screen host factors required for PEDV infection. Protein kinase C θ (PKCθ), a calcium-independent member of the PKC family localized in the cell membrane, was found to be a crucial host factor in PEDV infection. The investigation of PEDV infection was limited in Vero and porcine epithelial cell-jejunum 2 (IPEC-J2) due to defective interferon production in Vero and the poor replication of PEDV in IPEC-J2. Therefore, identifying suitable cells for PEDV investigation is crucial. The findings of this study reveal that human embryonic kidney (HEK) 293T and L929 cells, but not Vero and IPEC-J2 cells, were suitable for investigating PEDV infection. PKCθ played a significant role in endocytosis and the replication of PEDV, and PEDV regulated the expression and phosphorylation of PKCθ. Apoptosis was found to be involved in PEDV replication, as the virus activated the PKCθ-B-cell lymphoma 2 (BCL-2) ovarian killer (BOK) axis in HEK293T and L929 cells to increase viral endocytosis and replication via mitochondrial apoptosis. This study demonstrated the suitability of HEK293T and L929 cells for investigating PEDV infection and identified PKCθ as a host factor essential for PEDV infection. These findings provide valuable insights for the development of strategies and drug targets for PEDV infection.

The Peptidyl-Prolyl cis-trans isomerase, Pin1, associates with Protein Kinase C θ via a critical Phospho-Thr-Pro motif in the V3 regulatory domain.

Protein kinase C-θ (PKCθ) is a member of the novel PKC subfamily known for its selective and predominant expression in T lymphocytes where it regulates essential functions required for T cell activation and proliferation. Our previous studies provided a mechanistic explanation for the recruitment of PKCθ to the center of the immunological synapse (IS) by demonstrating that a proline-rich (PR) motif within the V3 region in the regulatory domain of PKCθ is necessary and sufficient for PKCθ IS localization and function. Herein, we highlight the importance of Thr335-Pro residue in the PR motif, the phosphorylation of which is key in the activation of PKCθ and its subsequent IS localization. We demonstrate that the phospho-Thr335-Pro motif serves as a putative binding site for the peptidyl-prolyl cis-trans isomerase (PPIase), Pin1, an enzyme that specifically recognizes peptide bonds at phospho-Ser/Thr-Pro motifs. Binding assays revealed that mutagenesis of PKCθ-Thr335-to-Ala abolished the ability of PKCθ to interact with Pin1, while Thr335 replacement by a Glu phosphomimetic, restored PKCθ binding to Pin1, suggesting that Pin1-PKCθ association is contingent upon the phosphorylation of the PKCθ-Thr335-Pro motif. Similarly, the Pin1 mutant, R17A, failed to associate with PKCθ, suggesting that the integrity of the Pin1 N-terminal WW domain is a requisite for Pin1-PKCθ interaction. In silico docking studies underpinned the role of critical residues in the Pin1-WW domain and the PKCθ phospho-Thr335-Pro motif, to form a stable interaction between Pin1 and PKCθ. Furthermore, TCR crosslinking in human Jurkat T cells and C57BL/6J mouse-derived splenic T cells promoted a rapid and transient formation of Pin1-PKCθ complexes, which followed a T cell activation-dependent temporal kinetic, suggesting a role for Pin1 in PKCθ-dependent early activation events in TCR-triggered T cells. PPIases that belong to other subfamilies, i.e., cyclophilin A or FK506-binding protein, failed to associate with PKCθ, indicating the specificity of the Pin1-PKCθ association. Fluorescent cell staining and imaging analyses demonstrated that TCR/CD3 triggering promotes the colocalization of PKCθ and Pin1 at the cell membrane. Furthermore, interaction of influenza hemagglutinin peptide (HA307-319)-specific T cells with antigen-fed antigen presenting cells (APCs) led to colocalization of PKCθ and Pin1 at the center of the IS. Together, we point to an uncovered function for the Thr335-Pro motif within the PKCθ-V3 regulatory domain to serve as a priming site for its activation upon phosphorylation and highlight its tenability to serve as a regulatory site for the Pin1 cis-trans isomerase.

LncRNA PRKCQ-AS1 regulates paclitaxel resistance in triple-negative breast cancer cells through miR-361-5p/PIK3C3 mediated autophagy.

Paclitaxel (PTX) resistance is a key cause of chemotherapy failure in patients with triple negative breast cancer (TNBC). The aim of this study is to investigate the effect and mechanism of long non-coding RNA (lncRNA) on the PTX resistance of TNBC cells through autophagy. MDA-MB-231 cells are used to induce the PTX-resistant TNBC cell line MDA-MB-231.PR (MDR) by increasing dose intermittently. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to validate the mRNA levels of phosphoinositide-3-kinase class 3 (PIK3C3), miR-361-5p and lncRNA PRKCQ-AS1 in the cells, and Western blot analysis was used to detect the protein expressions of PIK3C3, autophagy-related, drug-resistant and apoptosis-related genes. MDC staining detected the formation of autophagic vacuoles. The interactions between miR-361-5p and PIK3C3 and between lncRNA PRKCQ-AS1 and miR-361-5p were verified by dual-luciferase assay. Cell viability, apoptosis, migration and invasion were assessed by performing MTT, flow cytometry assay, and transwell assay. The mRNA level of miR-361-5p and the autophagy and drug resistance levels of TNBC PTX-resistant cells were significantly up-regulated. miR-361-5p could target autophagy-related gene PIK3C3, and overexpression of miR-361-5p could down-regulate PIK3C3 protein expression and autophagy level and PTX resistance of MDR cells. LncRNA PRKCQ-AS1 was selected through bioanalysis, and miR-361-5p could target lncRNA PRKCQ-AS1. In addition, lncRNA PRKCQ-AS1 level was up-regulated in TNBC PTX-resistant cells, and knockdown of lncRNA PRKCQ-AS1 could weaken autophagy and drug resistance level and could promote cell apoptosis. Overexpression of lncRNA PRKCQ-AS1 reversed the pro-apoptotic effect and down-regulation of autophagy and resistance levels was induced by miR-361-5p. In vivo experiments were performed to verify the role of lncRNA PRKCQ-AS1. We demonstrate that down-regulation of lncRNA PRKCQ-AS1 weakened PTX resistance and promoted cell apoptosis by miR-361-5p/PIK3C3 mediated autophagy.

Telomere-related gene risk model for prognosis and drug treatment efficiency prediction in kidney cancer.

Kidney cancer is one of the most common urological cancers worldwide, and kidney renal clear cell cancer (KIRC) is the major histologic subtype. Our previous study found that von-Hippel Lindau (VHL) gene mutation, the dominant reason for sporadic KIRC and hereditary kidney cancer-VHL syndrome, could affect VHL disease-related cancers development by inducing telomere shortening. However, the prognosis role of telomere-related genes in kidney cancer has not been well discussed. In this study, we obtained the telomere-related genes (TRGs) from TelNet. We obtained the clinical information and TRGs expression status of kidney cancer patients in The Cancer Genome Atlas (TCGA) database, The International Cancer Genome Consortium (ICGC) database, and the Clinical Proteomic Tumor Analysis Consortium (CPTAC) database. Totally 353 TRGs were differential between tumor and normal tissues in the TCGA-KIRC dataset. The total TCGA cohort was divided into discovery and validation TCGA cohorts and then using univariate cox regression, lasso regression, and multivariate cox regression method to conduct data analysis sequentially, ten TRGs (ISG15, RFC2, TRIM15, NEK6, PRKCQ, ATP1A1, ELOVL3, TUBB2B, PLCL1, NR1H3) risk model had been constructed finally. The kidney patients in the high TRGs risk group represented a worse outcome in the discovery TCGA cohort (p<0.001), and the result was validated by these four cohorts (validation TCGA cohort, total TCGA cohort, ICGC cohort, and CPTAC cohort). In addition, the TRGs risk score is an independent risk factor for kidney cancer in all these five cohorts. And the high TRGs risk group correlated with worse immune subtypes and higher tumor mutation burden in cancer tissues. In addition, the high TRGs risk group might benefit from receiving immune checkpoint inhibitors and targeted therapy agents. Moreover, the proteins NEK6, RF2, and ISG15 were upregulated in tumors both at the RNA and protein levels, while PLCL1 and PRKCQ were downregulated. The other five genes may display the contrary expression status at the RNA and protein levels. In conclusion, we have constructed a telomere-related genes risk model for predicting the outcomes of kidney cancer patients, and the model may be helpful in selecting treatment agents for kidney cancer patients.

Mapping brain gene coexpression in daytime transcriptomes unveils diurnal molecular networks and deciphers perturbation gene signatures.

Brain tissue transcriptomes may be organized into gene coexpression networks, but their underlying biological drivers remain incompletely understood. Here, we undertook a large-scale transcriptomic study using 508 wild-type mouse striatal tissue samples dissected exclusively in the afternoons to define 38 highly reproducible gene coexpression modules. We found that 13 and 11 modules are enriched in cell-type and molecular complex markers, respectively. Importantly, 18 modules are highly enriched in daily rhythmically expressed genes that peak or trough with distinct temporal kinetics, revealing the underlying biology of striatal diurnal gene networks. Moreover, the diurnal coexpression networks are a dominant feature of daytime transcriptomes in the mouse cortex. We next employed the striatal coexpression modules to decipher the striatal transcriptomic signatures from Huntington's disease models and heterozygous null mice for 52 genes, uncovering novel functions for Prkcq and Kdm4b in oligodendrocyte differentiation and bipolar disorder-associated Trank1 in regulating anxiety-like behaviors and nocturnal locomotion.

PLCγ1/PKCθ Downstream Signaling Controls Cutaneous T-Cell Lymphoma Development and Progression.

Developing mechanistic rationales can improve the clinical management of cutaneous T-cell lymphomas. There is considerable genetic and biological evidence of a malignant network of signaling mechanisms, highly influenced by deregulated TCR/PLCγ1 activity, controlling the biology of these lesions. In addition, activated signal transducer and activator of transcription 3 is associated with clinical progression, although the alterations responsible for this have not been fully elucidated. Here, we studied PLCγ1-dependent mechanisms that can mediate STAT3 activation and control tumor growth and progression. Downstream of PLCγ1, the pharmacological inhibition and genetic knockdown of protein kinase C theta (PKCθ) inhibited signal transducer and activator of transcription 3 activation, impaired proliferation, and promoted apoptosis in cutaneous T-cell lymphoma cells. A PKCθ-dependent transcriptome in mycosis fungoides/Sézary syndrome cells revealed potential effector genes controlling cytokine signaling, TP53, and actin cytoskeleton dynamics. Consistently, an in vivo chicken embryo model xenografted with mycosis fungoides cells showed that PKCθ blockage abrogates tumor growth and spread to distant organs. Finally, the expression of a number of PKCθ target genes found in mycosis fungoides cells significantly correlated with that of PRKCQ (PKCθ) in 81 human mycosis fungoides samples. In summary, PKCθ can play a central role in the activation of malignant cutaneous T-cell lymphoma mechanisms via multiple routes, including, but not restricted to, STAT3. These mechanisms may, in turn, serve as targets for specific therapies.

A 4 Gene-based Immune Signature Predicts Dedifferentiation and Immune Exhaustion in Thyroid Cancer.

CONTEXT: The role of immune-related genes (IRGs) in thyroid cancer dedifferentiation and accompanying immune exhaustion remains largely unexplored. OBJECTIVE: To construct a significant IRG-based signature indicative of dedifferentiation and immune exhaustion in thyroid cancer. DESIGN AND SETTINGS: One exploratory cohort and 2 validation cohorts were used to identify stably dysregulated IRGs in dedifferentiated thyroid cancer (DDTC) and to obtain independent risk factors for dedifferentiation. The IRGs formed a gene signature, whose predictive value was tested by the receiver operating characteristic curve. Correlations between the signature and differentiation-related genes, immune checkpoints, and prognosis were analyzed. Gene set enrichment analyses were performed to identify related signaling pathways. RESULTS: Four IRGs (PRKCQ, PLAUR, PSMD2, and BMP7) were found to be repeatedly dysregulated in DDTC, and they formed an IRG-based signature with a satisfactory predictive value for thyroid cancer dedifferentiation. Correlation analyses revealed that immune checkpoints were closely related to the 4 IRGs and the IRG-based signature, which was significantly associated with the histological subtype (P = 0.026), lymph node metastasis (P = 0.001), and BRAFV600E mutation (P < 0.001). The downregulated expression of PRKCQ shortened the disease-free survival for patients with thyroid cancer. Furthermore, we identified several signaling pathways inherently associated with the IRG-based signature. CONCLUSIONS: This study suggests that IRGs participate in the dedifferentiation and immune exhaustion process of thyroid cancer and are potential biomarkers for DDTC.

Protein kinase C-θ knockout decreases serum IL-10 levels and inhibits insulin secretion from islet ß cells.

Various subtypes of protein kinase C (PKC) are expressed in islet ß cells and regulate ß cell proliferation and survival. PKC-θ is distributed in the immune system and promotes the secretion of IL-10, which manifests a critical role in the onset of diabetes, by the immune cells. However, the role of PKC-θ in islets has not been concerned. In the present study, we investigated the role of PKC-θ in the protection of islet ß cells and insulin secretion. Fasting glucose and insulin measurement, glucose tolerant test, immunofluorescence, and ELISA were conducted to study the influence of PKC-θ knockout on islet ß cell survival and function, and explore the mechanism underlying this regulation. PKC-θ knockout mice at 2 weeks manifested normal serum insulin levels, glucose tolerance, and ß cell mass. Knockout mice at 8 weeks show decreased ß cell mass, but manifested normal insulin levels and glucose tolerance. Knockout mice at 16 weeks manifested impaired glucose tolerance, ß cell mass, and decreased glucose stimulated insulin secretion. Furthermore, knockout mice manifested decreased serum IL-10 level compared with normal mice since 2 weeks. IL-10 injection into knockout mice improved glucose tolerance, serum insulin level, and reduced ß cell mass, and IL-10 administration into cultured pancreatic tissue increased glucose stimulated insulin secretion. PKC-θ knockout decreases the secretion of IL-10, reduces ß cell mass and insulin secretion in pancreatic islets. The present study illuminates the critical role of PKC-θ in protecting the survival and function of islet ß cells.

Crosstalk between miR-203 and PKCθ regulates breast cancer stem cell markers.

INTRODUCTION: Protein kinase C theta (PKCθ) is expressed in ER-negative breast cancer and promotes cancer stem cells (CSCs) phenotype. PKCθ gene (PRKCQ) is predicted to be a target for tumor suppressor miR-203. Herein, we aim to validate this prediction and evaluate the ability of miR-203 to inhibit migration of breast cancer cell line enriched with CSCs, MDA-MB-231, via PRKCQ targeting. METHODS: Cells were transfected with miR-203 mimic, PRKCQ siRNA and negative control; then real-time PCR, migration assay, western blotting, reporter assay, and chromatin accessibility assay were performed. RESULTS: Our findings displayed significant decrease in PRKCQ mRNA level and luciferase signals in cells with restored miR-203 expression, therefore, validated PRKCQ as a direct target of miR-203. Additionally, inhibiting PRKCQ by siRNA led to significant inhibition of miR-203 expression and significant decrease of chromatin accessibility at miR-203 promoter region 466-291 upstream TSS. Both of miR-203 re-expression and PRKCQ suppression resulted in altering migration ability of MDA-MB-231 through regulating AKT pathway and genes involved in breast cancer stem cells, CD44 and ALDH1A3. Expression of CDK5, GIV, and NANOG was significantly downregulated in miR-203 mimic-transfected cells, while PRKCQ siRNA-transfected cells displayed downregulation of OCT3/4, SOX2, and NANOG. Furthermore, we found that miR-224 expression was enhanced while miR-150 was downregulated after ectopic expression of miR-203. CONCLUSION: The study highlighted the negative feedback loop between miR-203 and its target PRKCQ and the interplay between them in regulating genes involved in BCSCs. The study also concluded "microRNA-mediated microRNA regulation" as an event in breast cancer cells.

The Emerging Function of PKCtheta in Cancer.

Protein Kinase C theta (PKCθ) is a serine/threonine kinase that belongs to the novel PKC subfamily. In normal tissue, its expression is restricted to skeletal muscle cells, platelets and T lymphocytes in which PKCθ controls several essential cellular processes such as survival, proliferation and differentiation. Particularly, PKCθ has been extensively studied for its role in the immune system where its translocation to the immunological synapse plays a critical role in T cell activation. Beyond its physiological role in immune responses, increasing evidence implicates PKCθ in the pathology of various diseases, especially autoimmune disorders and cancers. In this review, we discuss the implication of PKCθ in various types of cancers and the PKCθ-mediated signaling events controlling cancer initiation and progression. In these types of cancers, the high PKCθ expression leads to aberrant cell proliferation, migration and invasion resulting in malignant phenotype. The recent development and application of PKCθ inhibitors in the context of autoimmune diseases could benefit the emergence of treatment for cancers in which PKCθ has been implicated.

Annexin A5 is essential for PKCθ translocation during T-cell activation.

T-cell activation is a critical part of the adaptive immune system, enabling responses to foreign cells and external stimulus. In this process, T-cell antigen receptor (TCR) activation stimulates translocation of the downstream kinase PKCθ to the membrane, leading to NF-κB activation and thus transcription of relevant genes. However, the details of how PKCθ is recruited to the membrane remain enigmatic. It is known that annexin A5 (ANXA5), a calcium-dependent membrane-binding protein, has been reported to mediate PKCδ activation by interaction with PKCδ, a homologue of PKCθ, which implicates a potential role of ANXA5 involved in PKCθ signaling. Here we demonstrate that ANXA5 does play a critical role in the recruitment of PKCθ to the membrane during T-cell activation. ANXA5 knockout in Jurkat T cells substantially inhibited the membrane translocation of PKCθ upon TCR engagement and blocked the recruitment of CARMA1-BCL10-MALT1 signalosome, which provides a platform for the catalytic activation of IKKs and subsequent activation of canonical NF-κB signaling in activated T cells. As a result, NF-κB activation was impaired in ANXA5-KO T cells. T-cell activation was also suppressed by ANAX5 knockdown in primary T cells. These results demonstrated a novel role of ANXA5 in PKC translocation and PKC signaling during T-cell activation.

PRKCQ inhibition enhances chemosensitivity of triple-negative breast cancer by regulating Bim.

BACKGROUND: Protein kinase C theta, (PRKCQ/PKCθ) is a serine/threonine kinase that is highly expressed in a subset of triple-negative breast cancers (TNBC) and promotes their growth, anoikis resistance, epithelial-mesenchymal transition (EMT), and invasion. Here, we show that PRKCQ regulates the sensitivity of TNBC cells to apoptosis triggered by standard-of-care chemotherapy by regulating levels of pro-apoptotic Bim. METHODS: To determine the effects of PRKCQ expression on chemotherapy-induced apoptosis, shRNA and cDNA vectors were used to modulate the PRKCQ expression in MCF-10A breast epithelial cells or triple-negative breast cancer cells (MDA-MB231Luc, HCC1806). A novel PRKCQ small-molecule inhibitor, 17k, was used to inhibit kinase activity. Viability and apoptosis of cells treated with PRKCQ cDNA/shRNA/inhibitor +/-chemotherapy were measured. Expression levels of Bcl2 family members were assessed. RESULTS: Enhanced expression of PRKCQ is sufficient to suppress apoptosis triggered by paclitaxel or doxorubicin treatment. Downregulation of PRKCQ also enhanced the apoptosis of chemotherapy-treated TNBC cells. Regulation of chemotherapy sensitivity by PRKCQ mechanistically occurs via regulation of levels of Bim, a pro-apoptotic Bcl2 family member; suppression of Bim prevents the enhanced apoptosis observed with combined PRKCQ downregulation and chemotherapy treatment. Regulation of Bim and chemotherapy sensitivity is significantly dependent on PRKCQ kinase activity; overexpression of a catalytically inactive PRKCQ does not suppress Bim or chemotherapy-associated apoptosis. Furthermore, PRKCQ kinase inhibitor treatment suppressed growth, increased anoikis and Bim expression, and enhanced apoptosis of chemotherapy-treated TNBC cells, phenocopying the effects of PRKCQ downregulation. CONCLUSIONS: These studies support PRKCQ inhibition as an attractive therapeutic strategy and complement to chemotherapy to inhibit the growth and survival of TNBC cells.

Long noncoding RNA PRKCQ-AS1 promotes CRC cell proliferation and migration via modulating miR-1287-5p/YBX1 axis.

Colorectal cancer (CRC) brings more than 600 000 deaths every year around the globe, making itself the third most frequently occurred carcinoma. The great progress human achieved in diagnosis and treatment of various cancers has failed to reverse this trend. Fortunately, growing evidence has implied the relationship between lncRNAs and cancer progression. Long noncoding RNA (lncRNA) PRKCQ-AS1 was heightened in CRC cells and tissues and related with dismal prognosis of CRC patients. Knockdown of PRKCQ-AS1 would induce a decrease in proliferative and migrating ability of CRC cells. Also, PRKCQ-AS1 enriched in cytoplasm of CRC cells and negatively regulated miR-1287-5p level. More important, PRKCQ-AS1 could bind to argonaute 2 and function in the RNA-induced silencing complex with miR-1287-5p. Therefore, PRKCQ-AS1 was a competing endogenous RNA for miR-1287-5p. Subsequently, it was validated that miR-1287-5p could suppress the proliferative and migratory functions in CRC. Furthermore, PRKCQ-AS1 could upregulate the mRNA and protein level of YBX1 targeted by miR-1287-5p. And YBX1 expression was elevated in CRC cells and tissues. Rescue assays in vitro and in vivo showed that overexpression of YBX1 could partly offset the effect of CRC progression induced by knocking down PRKCQ-AS1, demonstrating PRKCQ-AS1 mediating CRC progression via miR-1287-5p/YBX1 pathway.

Two novel pleiotropic loci associated with osteoporosis and abdominal obesity.

Aiming to uncover a shared genetic basis of abdominal obesity and osteoporosis, we performed a bivariate GWAS meta-analysis of femoral neck BMD (FNK-BMD) and trunk fat mass adjusted by trunk lean mass (TFMadj) in 11,496 subjects from 6 samples, followed by in silico replication in the large-scale UK Biobank (UKB) cohort. A series of functional investigations were conducted on the identified variants. Bivariate GWAS meta-analysis identified two novel pleiotropic loci 12q15 (lead SNP rs73134637, p = 3.45 × 10-7) and 10p14 (lead SNP rs2892347, p = 2.63 × 10-7) that were suggestively associated and that were replicated in the analyses of related traits in the UKB sample (osteoporosis p = 0.06 and 0.02, BMI p = 0.03 and 4.61 × 10-3, N up to 499,520). Cis-eQTL analysis demonstrated that allele C at rs73134637 was positively associated with IFNG expression in whole blood (N = 369, p = 0.04), and allele A at rs11254759 (10p14, p = 9.49 × 10-7) was negatively associated with PRKCQ expression in visceral adipose tissue (N = 313, p = 0.04) and in lymphocytes (N = 117, p = 0.03). As a proof-of-principle experiment, the function of rs11254759, which is 235 kb 5'-upstream from PRKCQ gene, was investigated by the dual-luciferase reporter assay, which clearly showed that the haplotype carrying rs11254759 regulated PRKCQ expression by upregulating PRKCQ promoter activity (p = 4.60 × 10-7) in an allelic specific manner. Mouse model analysis showed that heterozygous PRKCQ deficient mice presented decreased fat mass compared to wild-type control mice (p = 3.30 × 10-3). Mendelian randomization analysis demonstrated that both FNK-BMD and TFMadj were causally associated with fracture risk (p = 1.26 × 10-23 and 1.18 × 10-11). Our findings may provide useful insights into the genetic association between osteoporosis and abdominal obesity.

Targeting PKCθ Promotes Satellite Cell Self-Renewal.

Skeletal muscle regeneration following injury depends on the ability of satellite cells (SCs) to proliferate, self-renew, and eventually differentiate. The factors that regulate the process of self-renewal are poorly understood. In this study we examined the role of PKCθ in SC self-renewal and differentiation. We show that PKCθ is expressed in SCs, and its active form is localized to the chromosomes, centrosomes, and midbody during mitosis. Lack of PKCθ promotes SC symmetric self-renewal division by regulating Pard3 polarity protein localization, without affecting the overall proliferation rate. Genetic ablation of PKCθ or its pharmacological inhibition in vivo did not affect SC number in healthy muscle. By contrast, after induction of muscle injury, lack or inhibition of PKCθ resulted in a significant expansion of the quiescent SC pool. Finally, we show that lack of PKCθ does not alter the inflammatory milieu after acute injury in muscle, suggesting that the enhanced self-renewal ability of SCs in PKCθ-/- mice is not due to an alteration in the inflammatory milieu. Together, these results suggest that PKCθ plays an important role in SC self-renewal by stimulating their expansion through symmetric division, and it may represent a promising target to manipulate satellite cell self-renewal in pathological conditions.

Lack of PKCθ Promotes Regenerative Ability of Muscle Stem Cells in Chronic Muscle Injury.

Duchenne muscular dystrophy (DMD) is a genetic disease characterized by muscle wasting and chronic inflammation, leading to impaired satellite cells (SCs) function and exhaustion of their regenerative capacity. We previously showed that lack of PKCθ in mdx mice, a mouse model of DMD, reduces muscle wasting and inflammation, and improves muscle regeneration and performance at early stages of the disease. In this study, we show that muscle regeneration is boosted, and fibrosis reduced in mdxθ-/- mice, even at advanced stages of the disease. This phenotype was associated with a higher number of Pax7 positive cells in mdxθ-/- muscle compared with mdx muscle, during the progression of the disease. Moreover, the expression level of Pax7 and Notch1, the pivotal regulators of SCs self-renewal, were upregulated in SCs isolated from mdxθ-/- muscle compared with mdx derived SCs. Likewise, the expression of the Notch ligands Delta1 and Jagged1 was higher in mdxθ-/- muscle compared with mdx. The expression level of Delta1 and Jagged1 was also higher in PKCθ-/- muscle compared with WT muscle following acute injury. In addition, lack of PKCθ prolonged the survival and sustained the differentiation of transplanted myogenic progenitors. Overall, our results suggest that lack of PKCθ promotes muscle repair in dystrophic mice, supporting stem cells survival and maintenance through increased Delta-Notch signaling.

Exogenous carbon monoxide suppresses LPS-Induced platelet SNAREs complex assembly and α-granule exocytosis via integrin αIIbß3-Mediated PKCθ/Munc18a pathway.

Activation of coagulation occurs in sepsis and contributes to the development of thrombosis. Platelet α-granule exocytosis plays an important role in septic coagulation abnormalities. The present study aimed to investigate the effects and the underlying mechanisms of exogenous carbon monoxide, carbon monoxide-releasing molecules II (CORM-2)-liberated CO, on suppressing platelet α-granule exocytosis in sepsis. It was shown that CORM-2 weakened α-granule membrane fusion with platelet plasma membrane and attenuated α-granule contents exocytosis in LPS-Induced platelet. Further studies revealed that CORM-2 suppressed the expression of integrin αIIbß3 in platelets stimulated by LPS. This was accompanied by a decrease in production and phosphorylation of PKCθ and Munc18a, SNARE complex assembly and subsequently platelet α-granule exocytosis. Taken together, we suggested that the potential mechanism of suppressive effect of CORM-2 on LPS-induced platelet SNAREs complex assembly and α-Granule Exocytosis might involve integrin αIIbß3-mediated PKCθ/Munc18a pathway activation.

Loss-of-function phenotype of a PKCθT219A knockin mouse strain.

BACKGROUND: Protein kinase C θ has been established as an important signaling intermediate in T-effector-cell activation and survival pathways by controlling activity of the key transcription factors NF-κB and NFAT. Previous studies identified an activation-induced auto-phosphorylation site at Thr-219, located between the tandem C1 domains of the regulatory fragment in PKCθ, as a structural requirement for its correct membrane translocation and the subsequent transactivation of downstream signals leading to IL-2 production in a human T cell line. METHODS: The present work aimed to define the role of this phosphorylation switch on PKCθ in a physiological context through a homozygous T219A knockin mouse strain. T cell activation was analyzed by H3-thymidine uptake (proliferative response), qRT-PCR and luminex measurements (cytokine production). NFAT and NF-κB transactivation responses were estimated by Gel mobility shift and Alpha Screen assays. Frequencies of T cell subsets were analyzed by flow cytometry. RESULTS: Despite a normal T cell development, in vitro activated effector T cells clearly revealed a requirement of Thr-219 phosphorylation site on PKCθ for a transactivation of NF-κB and NFAT transcription factors and, subsequently, robust IL-2 and IFN-γ expression. CONCLUSION: This phenotype is reminiscent of the PKCθ knockout T cells, physiologically validating that this (p) Thr-219 auto-phosphorylation site indeed critically regulates PKCθ function in primary mouse T cells.

Novel mutant mouse line emphasizes the importance of protein kinase C theta for CD4+ T lymphocyte activation.

BACKGROUND: The protein kinase C theta (PKCθ) has an important and non-redundant function downstream of the antigen receptor and co-receptor complex in T lymphocytes. PKCθ is not only essential for activation of NF-κB, AP-1 and NFAT and subsequent interleukin-2 expression, but also critical for positive selection and development of regulatory T lymphocytes in the thymus. Several domains regulate its activity, such as a pseudosubstrate sequence mediating an auto-inhibitory intramolecular interaction, the tandem C1 domains binding diacylglycerol, and phosphorylation at conserved tyrosine, threonine as well as serine residues throughout the whole length of the protein. To address the importance of the variable domain V1 at the very N-terminus, which is encoded by exon 2, a mutated version of PKCθ was analyzed for its ability to stimulate T lymphocyte activation. METHODS: T cell responses were analyzed with promoter luciferase reporter assays in Jurkat T cells transfected with PKCθ expression constructs. A mouse line expressing mutated instead of wild type PKCθ was analyzed in comparison to PKCθ-deficient and wild type mice for thymic development and T cell subsets by flow cytometry and T cell activation by quantitative RT-PCR, luminex analysis and flow cytometry. RESULTS: In cell lines, the exon 2-replacing mutation impaired the transactivation of interleukin-2 expression by constitutively active mutant form of PKCθ. Moreover, analysis of a newly generated exon 2-mutant mouse line (PKCθ-E2mut) revealed that the N-terminal replacement mutation results in an hypomorph mutant of PKCθ combined with reduced PKCθ protein levels in CD4+ T lymphocytes. Thus, PKCθ-dependent functions in T lymphocytes were affected resulting in impaired thymic development of single positive T lymphocytes in vivo. In particular, there was diminished generation of regulatory T lymphocytes. Furthermore, early activation responses such as interleukin-2 expression of CD4+ T lymphocytes were significantly reduced even though cell viability was not affected. Thus, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. CONCLUSION: Taken together, PKCθ-E2mut mice show a phenotype similar to conventional PKCθ-deficient mice. Both our in vitro T cell culture experiments and ex vivo analyses of a PKCθ-E2-mutant mouse line independently validate the importance of PKCθ downstream of the antigen-receptor complex for activation of CD4+ T lymphocytes.

Phosphotyrosine-dependent interaction between the kinases PKCθ and Zap70 promotes proximal TCR signaling.

Protein kinase C-θ (PKCθ) is an important component of proximal T cell receptor (TCR) signaling. We previously identified the amino-terminal C2 domain of PKCθ as a phosphotyrosine (pTyr)-binding domain. Using a mutant form of PKCθ that cannot bind pTyr (PKCθHR2A), we showed that pTyr binding by PKCθ was required for TCR-induced T cell activation, proliferation, and TH2 cell differentiation but not for T cell development. Using tandem mass spectrometry and coimmunoprecipitation, we identified the kinase ζ-associated protein kinase of 70 kDa (Zap70) as a binding partner of the PKCθ pTyr-binding pocket. Tyr126 of Zap70 directly bound to PKCθ, and the interdomain B residues Tyr315 and Tyr319 were indirectly required for binding to PKCθ, reflecting their role in promoting the open conformation of Zap70. PKCθHR2A-expressing CD4+ T cells displayed defects not only in known PKCθ-dependent signaling events, such as nuclear factor κB (NF-κB) activation and TH2 cell differentiation, but also in full activation of Zap70 itself and in the activating phosphorylation of linker of activation of T cells (LAT) and phospholipase C-γ1 (PLCγ1), signaling proteins that are traditionally considered to be activated independently of PKC. These findings demonstrate that PKCθ plays an important role in a positive feedback regulatory loop that modulates TCR-proximal signaling and, moreover, provide a mechanistic explanation for earlier reports that documented an important role for PKCθ in T cell Ca2+ signaling. This PKCθ-Zap70 interaction could potentially serve as a promising and highly selective immunosuppressive drug target in autoimmunity and organ transplantation.