The role of protein kinase D (PKD) in obesity: Lessons from the heart and other tissues.

Obesity causes a range of tissue dysfunctions that increases the risk for morbidity and mortality. Protein kinase D (PKD) represents a family of stress-activated intracellular signalling proteins that regulate essential processes such as cell proliferation and differentiation, cell survival, and exocytosis. Evidence suggests that PKD regulates the cellular adaptations to the obese environment in metabolically important tissues and drives the development of a variety of diseases. This review explores the role that PKD plays in tissue dysfunction in obesity, with special consideration of the development of obesity-mediated cardiomyopathy, a distinct cardiovascular disease that occurs in the absence of common comorbidities and leads to eventual heart failure and death. The downstream mechanisms mediated by PKD that could contribute to dysfunctions observed in the heart and other metabolically important tissues in obesity, and the predicted cell types involved are discussed to suggest potential targets for the development of therapeutics against obesity-related disease.

Pharmacological inhibition of protein kinase D2/Aurora kinase A signalling axis suppresses G2/M cell cycle progression and proliferation of epithelial ovarian cancer cells.

Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy with poor prognosis and patient survival outcome. Protein kinase D2 (PKD2) belongs to Ca++/calmodulin-dependent serine/threonine kinase family and its aberrant expression is associated with many cellular and physiological functions associated with tumorigenesis including cell proliferation. We show that PKD2 is activated during G2/M cell cycle transition and its catalytic inactivation by small molecule inhibitor CRT0066101 or genetic knockdown caused suppression of EOC cell proliferation followed by a delay into mitotic entry. Our RNASeq analysis of PKD2-inactivated EOC cells revealed significant downregulation of genes associated with cell cycle including Aurora kinase A, a critical mitotic regulator. Mechanistically, PKD2 positively regulated Aurora kinase A stability at both transcriptional and post-translational levels by interfering with the function of Fbxw7, drove G2/M cell cycle transition and EOC cell proliferation. Moreover, pharmacological inhibition of Aurora kinase A by small molecule CD532 or its shRNA-mediated genetic knockdown suppressed EOC cell proliferation, induced G2/M cell cycle arrest and mitotic catastrophe followed by apoptosis. Taken together, our results indicated that PKD2 positively regulates Aurora kinase A during G2/M cell cycle entry and pharmacological targeting of PKD2/Aurora kinase A signalling axis could serve as a novel therapeutic intervention against a lethal pathology like EOC.

Natural variation in protein kinase D modifies alcohol sensitivity in Caenorhabditis elegans.

Differences in naïve alcohol sensitivity between individuals are a strong predictor of later life alcohol use disorders (AUD). However, the genetic bases for alcohol sensitivity (beyond ethanol metabolism) and pharmacological approaches to modulate alcohol sensitivity remain poorly understood. We used a high-throughput behavioral screen to measure acute behavioral sensitivity to alcohol, a model of intoxication, in a genetically diverse set of over 150 wild strains of the nematode Caenorhabditis elegans. We performed a genome-wide association study to identify loci that underlie natural variation in alcohol sensitivity. We identified five quantitative trait loci (QTL) and further show that variants in the C. elegans ortholog of protein kinase D, dkf-2, likely underlie the chromosome V QTL. We found that resistance to intoxication was conferred by dkf-2 loss-of-function mutations as well as partly by a PKD inhibitor in a dkf-2-dependent manner. Protein kinase D might represent a conserved, druggable target to modify alcohol sensitivity with application towards AUD.

Protein kinase-D1 and downstream signaling mechanisms involved in GLUT4 translocation in cardiac muscle.

The Ser/Thr kinase protein kinase-D1 (PKD1) is involved in induction of various cell physiological processes in the heart such as myocellular hypertrophy and inflammation, which may turn maladaptive during long-term stimulation. Of special interest is a key role of PKD1 in the regulation of cardiac substrate metabolism. Glucose and fatty acids are the most important substrates for cardiac energy provision, and the ratio at which they are utilized determines the health status of the heart. Cardiac glucose uptake is mainly regulated by translocation of the glucose transporter GLUT4 from intracellular stores (endosomes) to the sarcolemma, and fatty acid uptake via a parallel translocation of fatty acid transporter CD36 from endosomes to the sarcolemma. PKD1 is involved in the regulation of GLUT4 translocation, but not CD36 translocation, giving it the ability to modulate glucose uptake without affecting fatty acid uptake, thereby altering the cardiac substrate balance. PKD1 would therefore serve as an attractive target to combat cardiac metabolic diseases with a tilted substrate balance, such as diabetic cardiomyopathy. However, PKD1 activation also elicits cardiac hypertrophy and inflammation. Therefore, identification of the events upstream and downstream of PKD1 may provide superior therapeutic targets to alter the cardiac substrate balance. Recent studies have identified the lipid kinase phosphatidylinositol 4-kinase IIIß (PI4KIIIß) as signaling hub downstream of PKD1 to selectively stimulate GLUT4-mediated myocardial glucose uptake without inducing hypertrophy. Taken together, the PKD1 signaling pathway serves a pivotal role in cardiac glucose metabolism and is a promising target to selectively modulate glucose uptake in cardiac disease.

Protein kinase D2-Aurora kinase A-ERK1/2 signalling axis drives neuroendocrine differentiation of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is deadliest gynecological malignancy with poor prognosis and patient survival. Despite development of several therapeutic interventions such as poly-ADP ribose polymerase (PARP) inhibitors, EOC remains unmanageable and discovery of novel early detection biomarkers and treatment targets are highly warranted. Although neuroendocrine differentiation (NED) is implicated in different human cancers including prostate adenocarcinoma and lung cancer, mechanistic studies concerning NED of epithelial ovarian cancer are lacking. We report that Aurora kinase A drives NED of epithelial ovarian cancer in an ERK1/2-dependent manner and pharmacological and genetic inhibition of Aurora kinase A suppress NED of ovarian cancer. Moreover, we demonstrate that protein kinase D2 positively regulated Aurora kinase A to drive NED. Overexpression of catalytically active PKD2 drives NED and collectively, PKD2 cross talks with Aurora kinase A/ERK1/2 signalling axis to positively regulate NED of EOC. PKD2/Aurora kinase A/ERK1/2 signalling axis is a novel therapeutic target against neuroendocrine differentiated EOC.

Adeno-associated virus-based approach for genetic modification of cardiac fibroblasts in adult rat hearts.

Cardiac fibroblasts (CFs) are an attractive target for reducing pathological cardiac remodeling, and understanding the underlying mechanisms of these processes is the key to develop successful therapies for treating the pressure-overloaded heart. CF-specific knockout (KO) mouse lines with a Cre recombinase under the control of human TCF21 (hTCF21) promoter and/or an adeno-associated virus serotype 9 (AAV9)-hTCF21 system provide a powerful tool for understanding CF biology in vivo. Although a variety of rat disease models are vital for the research of cardiac fibrosis similar to mouse models, there are few rat models that employ cardiac cell-specific conditional gene modification, which has hindered the development and translational relevance of cardiac disease models. In addition, to date, there are no reports of gene manipulation specifically in rat CFs in vivo. Here, we report a simplified CF-specific rat transgenic model using an AAV9-hTCF21 system that achieved a CF-specific expression of transgene in adult rat hearts. Moreover, we successfully applied this approach to specifically manipulate mitochondrial morphology in quiescent CFs. In summary, this model will allow us to develop fast and simple rat CF-specific transgenic models for studying cardiovascular diseases in vivo.

Protein Kinase D Plays a Crucial Role in Maintaining Cardiac Homeostasis by Regulating Post-Translational Modifications of Myofilament Proteins.

Protein kinase D (PKD) enzymes play important roles in regulating myocardial contraction, hypertrophy, and remodeling. One of the proteins phosphorylated by PKD is titin, which is involved in myofilament function. In this study, we aimed to investigate the role of PKD in cardiomyocyte function under conditions of oxidative stress. To do this, we used mice with a cardiomyocyte-specific knock-out of Prkd1, which encodes PKD1 (Prkd1loxP/loxP; αMHC-Cre; PKD1 cKO), as well as wild type littermate controls (Prkd1loxP/loxP; WT). We isolated permeabilized cardiomyocytes from PKD1 cKO mice and found that they exhibited increased passive stiffness (Fpassive), which was associated with increased oxidation of titin, but showed no change in titin ubiquitination. Additionally, the PKD1 cKO mice showed increased myofilament calcium (Ca2+) sensitivity (pCa50) and reduced maximum Ca2+-activated tension. These changes were accompanied by increased oxidation and reduced phosphorylation of the small myofilament protein cardiac myosin binding protein C (cMyBPC), as well as altered phosphorylation levels at different phosphosites in troponin I (TnI). The increased Fpassive and pCa50, and the reduced maximum Ca2+-activated tension were reversed when we treated the isolated permeabilized cardiomyocytes with reduced glutathione (GSH). This indicated that myofilament protein oxidation contributes to cardiomyocyte dysfunction. Furthermore, the PKD1 cKO mice exhibited increased oxidative stress and increased expression of pro-inflammatory markers interleukin (IL)-6, IL-18, and tumor necrosis factor alpha (TNF-α). Both oxidative stress and inflammation contributed to an increase in microtubule-associated protein 1 light chain 3 (LC3)-II levels and heat shock response by inhibiting the mammalian target of rapamycin (mTOR) in the PKD1 cKO mouse myocytes. These findings revealed a previously unknown role for PKD1 in regulating diastolic passive properties, myofilament Ca2+ sensitivity, and maximum Ca2+-activated tension under conditions of oxidative stress. Finally, we emphasized the importance of PKD1 in maintaining the balance of oxidative stress and inflammation in the context of autophagy, as well as cardiomyocyte function.

Effect of deletion of the protein kinase PRKD1 on development of the mouse embryonic heart.

Congenital heart disease (CHD) is the most common congenital anomaly, with an overall incidence of approximately 1% in the United Kingdom. Exome sequencing in large CHD cohorts has been performed to provide insights into the genetic aetiology of CHD. This includes a study of 1891 probands by our group in collaboration with others, which identified three novel genes-CDK13, PRKD1, and CHD4, in patients with syndromic CHD. PRKD1 encodes a serine/threonine protein kinase, which is important in a variety of fundamental cellular functions. Individuals with a heterozygous mutation in PRKD1 may have facial dysmorphism, ectodermal dysplasia and may have CHDs such as pulmonary stenosis, atrioventricular septal defects, coarctation of the aorta and bicuspid aortic valve. To obtain a greater appreciation for the role that this essential protein kinase plays in cardiogenesis and CHD, we have analysed a Prkd1 transgenic mouse model (Prkd1em1) carrying deletion of exon 2, causing loss of function. High-resolution episcopic microscopy affords detailed morphological 3D analysis of the developing heart and provides evidence for an essential role of Prkd1 in both normal cardiac development and CHD. We show that homozygous deletion of Prkd1 is associated with complex forms of CHD such as atrioventricular septal defects, and bicuspid aortic and pulmonary valves, and is lethal. Even in heterozygotes, cardiac differences occur. However, given that 97% of Prkd1 heterozygous mice display normal heart development, it is likely that one normal allele is sufficient, with the defects seen most likely to represent sporadic events. Moreover, mRNA and protein expression levels were investigated by RT-qPCR and western immunoblotting, respectively. A significant reduction in Prkd1 mRNA levels was seen in homozygotes, but not heterozygotes, compared to WT littermates. While a trend towards lower PRKD1 protein expression was seen in the heterozygotes, the difference was only significant in the homozygotes. There was no compensation by the related Prkd2 and Prkd3 at transcript level, as evidenced by RT-qPCR. Overall, we demonstrate a vital role of Prkd1 in heart development and the aetiology of CHD.

Role of protein kinase D1 in vasoconstriction and haemodynamics in rats.

AIMS: Protein kinase D (PKD), once considered an effector of protein kinase C (PKC), now plays many pathophysiological roles in various tissues. However, little is known about role of PKD in vascular function. We investigated the role of PKD in contraction of rat aorta and human aortic smooth muscle cells (HASMCs) and in haemodynamics in rats. METHODS AND RESULTS: Isometric tension of rat aortic was measured to examine norepinephrine-induced contraction in the presence of PKD, PKC and Rho-kinase inhibitors. Phosphorylation of PKD1, myosin targeting subunit-1 (MYPT1), myosin light chain (MLC), CPI-17 and heat-shock protein 27 (HSP27), and actin polymerization were measured in the aorta. Phosphorylation of MYPT1 and MLC was also measured in HASMCs knocked down with specific siRNAs of PKD 1, 2 and 3. Intracellular calcium concentrations and cell shortening were measured in HASMCs. Norepinephrine-induced aortic contraction was accompanied by increased phosphorylation of PKD1, MYPT1 and MLC and actin polymerization, all of which were attenuated with PKD inhibitor CRT0066101. PKD1 phosphorylation was not inhibited by PKC inhibitor, chelerythrine or Rho kinase inhibitor, fasudil. In HASMCs, the phosphorylation of MYPT1 and MLC was attenuated by PKD1, but not PKD2, 3 knockdown. In HASMCs, CRT0066101 inhibited norepinephrine-induced cell shortening without affecting calcium concentration. Administration of CRT0066101 decreased systemic vascular resistance and blood pressure without affecting cardiac output in rats. CONCLUSIONS: PKD1 may play roles in aorta contraction and haemodynamics via phosphorylation of MYPT1 and actin polymerization in a calcium-independent manner.

Targeting PKD2 aggravates ferritinophagy-mediated ferroptosis via promoting autophagosome-lysosome fusion and enhances efficacy of carboplatin in lung adenocarcinoma.

Ferroptosis is an iron-dependent cell death and affects efficacies of multiple antitumor regimens, showing a great potential in cancer therapy. Protein kinase D2 (PKD2) plays a crucial role in regulating necrosis and apoptosis. However, the relationship of PKD2 and ferroptosis is still elusive. In this study, we mainly analyzed the roles of PKD2 on ferroptosis and chemotherapy in lung adenocarcinoma (LUAD). We found PKD2 was highly expressed in LUAD and silencing PKD2 could promote erastin-induced reactive oxygen species (ROS), malondialdehyde (MDA) accumulation, intracellular iron content and LUAD cells death. Mechanistically, augmenting PKD2 could prevent autophagic degradation of ferritin, which could be impaired by bafilomycin A1. We further found that PKD2 overexpression would promote LC3B-II, p62/SQSTM1 accumulation and block autophagosome-lysosome fusion in a TFEB-independent manner, which could be impaired by bafilomycin A1. Bafilomycin A1 stimulation could weaken ferroptosis promotion by PKD2 abrogation. Silencing ferritin heavy chain-1 (FTH1) could reverse the resistance to ferroptosis by PKD2 overexpression. Additionally, in vitro and vivo experiments validated PKD2 promoted proliferation, migration and invasion of LUAD cells. PKD2 knockdown or pharmacological inhibition by CRT0066101 could enhance efficacy of carboplatin in LUAD via ferroptosis and apoptosis. Collectively, our study revealed that abrogation of PKD2 could aggravate ferritinophagy-mediated ferroptosis by promoting autophagosome-lysosome fusion and enhance efficacy of carboplatin in LUAD. Targeting PKD2 to induce ferroptosis may be a promising strategy for LUAD therapy.

Protein kinase D1 - A targetable mediator of pancreatic cancer development.

Members of the Protein kinase D (PKD) kinase family each play important cell-specific roles in the regulation of normal pancreas functions. In pancreatic diseases PKD1 is the most widely characterized isoform with roles in pancreatitis and in induction of pancreatic cancer and its progression. PKD1 expression and activation increases in pancreatic acinar cells through macrophage secreted factors, Kirsten rat sarcoma viral oncogene homolog (KRAS) signaling, and reactive oxygen species (ROS), driving the formation of precancerous lesions. In precancerous lesions PKD1 regulates cell survival, growth, senescence, and generation of doublecortin like kinase 1 (DCLK1)-positive cancer stem cells (CSCs). Within tumors, regulation by PKD1 includes chemoresistance, apoptosis, proliferation, CSC features, and the Warburg effect. Thus, PKD1 plays a critical role throughout pancreatic disease initiation and progression.

Crosstalk between P2Y receptors and cyclooxygenase activity in inflammation and tissue repair.

The role of extracellular nucleotides as modulators of inflammation and cell stress is well established. One of the main actions of these molecules is mediated by the activation of purinergic receptors (P2) of the plasma membrane. P2 receptors can be classified according to two different structural families: P2X ionotropic ion channel receptors, and P2Y metabotropic G protein-coupled receptors. During inflammation, damaged cells release nucleotides and purinergic signaling occurs along the temporal pattern of the synthesis of pro-inflammatory and pro-resolving mediators by myeloid and lymphoid cells. In macrophages under pro-inflammatory conditions, the expression and activity of cyclooxygenase 2 significantly increases and enhances the circulating levels of prostaglandin E2 (PGE2), which exerts its effects both through specific plasma membrane receptors (EP1-EP4) and by activation of intracellular targets. Here we review the mechanisms involved in the crosstalk between PGE2 and P2Y receptors on macrophages, which is dependent on several isoforms of protein kinase C and protein kinase D1. Due to this crosstalk, a P2Y-dependent increase in calcium is blunted by PGE2 whereas, under these conditions, macrophages exhibit reduced migratory capacity along with enhanced phagocytosis, which contributes to the modulation of the inflammatory response and tissue repair.

The novel mechanism of human norovirus induced diarrhea: Activation of PKD2 caused by HuNoVs destroyed AQP3 expression through AP2γ in intestinal epithelial cells.

Our previous work has demonstrated protein kinase D2 (PKD2) played a critical influence in experimental colitis in animal. However, the role of PKD2 in human norovirus (HuNoVs)-induced diarrhea remained unknown. Aquaporin 3 (AQP3) expression, a critical protein mediating diarrhea, was assessed by western blot, qRT-PCR in intestinal epithelial cells (IECs). Luciferase, IF, IP and ChIP assay were used to explore the mechanism through which HuNoVs regulated AQP3. Herein, we found that AQP3 expression was drastically decreased in IECs in response to VP1 transfection, the major capsid protein of HuNoVs, or HuNoVs infection. Mechanistically, HuNoVs triggered phosphorylation of PKD2 through TLR2/MyD88/IRAK4, which further inhibited AP2γ activation and nuclear translocation, leading to suppress AQP3 transactivation in IECs. Most importantly, PKD2 interacted with MyD88/IRAK4, and VP1 overexpression enhanced this complex form, which, in turn, to increase PKD2 phosphorylation. In addition, endogenous PKD2 interacted with AP2γ, and this interaction was enhanced in response to HuNoVs treatment, and subsequently resulting in AP2γ phosphorylation inhibition. Moreover, inhibition of PKD2 activation could reverse the inhibitory effect of HuNoVs on AQP3 expression. In summary, we established a novel mechanism that HuNoV inhibited AQP3 expression through TLR2/MyD88/IRAK4/PKD2 signaling pathway, targeting PKD2 activity could be a promising strategy for prevention of HuNoVs-induced gastroenteritis.

The role of PKC and PKD in CXCL12 and CXCL13 directed malignant melanoma and acute monocytic leukemic cancer cell migration.

Cancer metastasis is the leading cause of cancer related mortality. Chemokine receptors and proteins in their downstream signalling axis represent desirable therapeutic targets for the prevention of metastasis. Despite this, current therapeutics have experienced limited success in clinical trials due to a lack of insight into the downstream signalling pathway of specific chemokine receptor cascades in different tumours. In this study, we investigated the role of protein kinase C (PKC) and protein kinase D (PKD) in CXCL12 and CXCL13 stimulated SK-MEL-28 (malignant melanoma) and THP-1 (acute monocytic leukaemia) cell migration. While PKC and PKD had no active role in CXCL12 or CXCL13 stimulated THP-1 cell migration, PKC and PKD inhibition reduced CXCL12 stimulated migration and caused profound effects upon the cytoskeleton of SK-MEL-28 cells. Furthermore, only PKC and not PKD inhibition reduced CXCL13 stimulated migration in SK-MEL-28 cells however PKC inhibition failed to stimulate any changes to the actin cytoskeleton. These findings indicate that PKC inhibitors would be a useful therapeutic for the prevention of both CXCL12 and CXCL13 stimulated migration and PKD inhibitors for CXCL12 stimulated migration in malignant melanoma.

PKD knockdown mitigates Ang II-induced cardiac hypertrophy and ferroptosis via the JNK/P53 signaling pathway.

BACKGROUND: Cardiac hypertrophy is studied in relation to energy metabolism, autophagy, and ferroptosis, which are associated with cardiovascular adverse events and chronic heart failure. Protein kinase D (PKD) has been shown to play a degenerative role in cardiac hypertrophy. However, the role of ferroptosis in PKD-involved cardiac hypertrophy remains unclear. METHODS: A cardiac hypertrophy model was induced by a subcutaneous injection of angiotensin II (Ang II) for 4 weeks. Adeno-associated virus serotype 9 (AAV9)-PKD or AAV9-Negative control were injected through the caudal vein 2 weeks prior to the injection of Ang II. The degree of cardiac hypertrophy was assessed using echocardiography and by observing cardiomyocyte morphology. Levels of ferroptosis and protein expression in the Jun N-terminal kinase (JNK)/P53 signaling pathway were measured both in vivo and in vitro. RESULTS: The results indicated that PKD knockdown reduces Ang II-induced cardiac hypertrophy, enhances cardiac function and inhibits ferroptosis. The involvement of the JNK/P53 pathway in this process was further confirmed by in vivo and in vitro experiments. CONCLUSION: In conclusion, our findings suggest that PKD knockdown mitigates Ang II-induced cardiac hypertrophy and ferroptosis via the JNK/P53 signaling pathway.

Protein kinase D (PKD) on the crossroad of lipid absorption, synthesis and utilization.

Inappropriate lipid levels in the blood, as well as its content and composition in different organs, underlie multiple metabolic disorders including obesity, non-alcoholic fatty liver disease, type 2 diabetes, and atherosclerosis. Multiple processes contribute to the complex metabolism of triglycerides (TGs), fatty acids (FAs), and other lipid species. These consist of digestion and absorption of dietary lipids, de novo FAs synthesis (lipogenesis), uptake of TGs and FAs by peripheral tissues, TGs storage in the intracellular depots as well as lipid utilization for ß-oxidation and their conversion to lipid-derivatives. A majority of the enzymatic reactions linked to lipogenesis, TGs synthesis, lipid absorption, and transport are happening at the endoplasmic reticulum, while ß-oxidation takes place in mitochondria and peroxisomes. The Golgi apparatus is a central sorting, protein- and lipid-modifying organelle and hence is involved in lipid metabolism as well. However, the impact of the processes taking part in the Golgi apparatus are often overseen. The protein kinase D (PKD) family (composed of three members, PKD1, 2, and 3) is the master regulator of Golgi dynamics. PKDs are also a sensor of different lipid species in distinct cellular compartments. In this review, we discuss the roles of PKD family members in the regulation of lipid metabolism including the processes executed by PKDs at the Golgi apparatus. We also discuss the role of PKDs-dependent signaling in different cellular compartments and organs in the context of the development of metabolic disorders.

Improvement effect of ligustilide on rats with heart failure by regulating PKD1/HIF-1α/VEGF pathway / 吉林大学学报(医学版)

Objective:To discuss the effect of ligustilide on the cardiac function and angiogenesis in the rats with heart failure,and to clarify its regulatory effect on protein kinase D1(PKD1)/hypoxia-inducible factor-1α(HIF-1α)/vascular endothelial growth factor(VEGF)pathway.Methods:The SD rats were randomly divided into sham operation group,model group,ligustilide group,PKD1/HIF-1α/VEGF signaling pathway inhibitor CID755673(CID)group,and ligustilide+CID group.The heart failure rat model was established by ligation of the left anterior descending coronary artery.The rats in ligustilide group were injected intravenously with 20 mg·kg-1 ligustilide,the rats in CID group were injected intraperitoneally with 50 mg·kg-1 CID,and the rats in ligustilide+CID group were injected intraperitoneally with 50 mg·kg-1 CID followed by intravenous injection of 20 mg·kg-1 ligustilide,once per day for 4 consecutive weeks.The cardiac function indexes of the rats in various groups were detected by echocardiography;the percentages of myocardial infarction areas of the rats in various groups were detected by 2,3,5-triphenyltetrazolium chloride(TTC)staining;the pathomorphology of myocardium tissue of the rats in various groups was observed by HE staining;the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in ischemic area of myocardium tissue of the rats in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR)and Western blotting methods.Results:Compared with sham operation group,the rats in model group and CID group had altered myocardial cell morphology,increased intercellular gaps,disorganized arrangement,visible muscle fiber breaks and inflammatory cell infiltration;the rats in ligustilide group and ligustilide+CID group had relatively orderly myocardial fiber arrangement,fewer myocardial fiber breaks and decreased number of inflammatory cells.Compared with sham operation group,the left ventricular ejection fraction(LVEF)and left ventricular fractional shortening(LVFS)of the rats in model group were decreased(P<0.05),the left ventricular end-systolic diameter(LVESD)and left ventricular end-diastolic diameter(LVEDD)were increased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were decreased(P<0.05).Compared with model group,the LVEF and LVFS of the rats in ligustilide group were increased(P<0.05),the LVESD and LVEDD were decreased(P<0.05),the percentage of myocardium infarction area was decreased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were increased(P<0.05);compared with model group,the LVEF and LVFS of the rats in CID group were decreased(P<0.05),the LVESD and LVEDD were increased(P<0.05),the percentage of myocardium infarction area was increased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were decreased(P<0.05);compared with ligustilide group,the LVEF and LVFS of the rats in ligustilide+CID group were decreased(P<0.05),the LVESD and LVEDD were increased(P<0.05),the percentage of myocardium infarction area was increased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were decreased(P<0.05);compared with CID group,the LVEF and LVFS of the rats in ligustilide+CID group were increased(P<0.05),the LVESD and LVEDD were decreased(P<0.05),the percentage of myocardium infarction area was decreased(P<0.05),and the expression levels of PKD1,HIF-1α,CD31,and VEGF mRNA and proteins in myocardium tissue were increased(P<0.05).Conclusion:Ligustilide can promote the angiogenesis,reduce the myocardium infarction area,and improve the cardiac function in the rats with heart failure;it works through activation of the PKD1/HIF-1α/VEGF pathway.

Mechanism of FTO-mediated and m6A-modified PRKD2 Inhibiting Podocyte Injury in Diabetic Kidney Disease through the SIRT1/HIF-1 Pathway / 现代检验医学杂志

Objective To explore the regulatory role of fat mass and obesity-associated protein(FTO)and serine-threonine kinase protein kinase D2(PRKD2)in progression of diabetic kidney disease(DKD)and its regulatory mechanisms.Methods DKD model in vitro was constructed by podocytes(MPC5 cells)treated with high glucose(HG,35 mmol/L glucose)for 24 h.HG-induced MPC5 cells were transfected with FTO overexpression vector(pcDNA-FTO)and PRKD2 overexpression vector(pcDNA-PRKD2),or empty vector.The overexpression efficiency of FTO and PRKD2 were detected with RT-qPCR.MeRIP was used to detect the N6-methyladenosine(m6A)modification level of PRKD2 mRNA.The activity of Caspase-3 and the secretion of IL-6,TNF-α and monocyte chemotactic protein-1(MCP-1)were detected by ELISA.Cell apoptosis rate was analyzed by flow cytometry.The protein levels of FTO and PRKD2,as well as the key proteins in SIRT1/HIF-1α pathway,were evaluated by Western blot.Pearson analysis was used to analyze the correlation between FTO levels and PRKD2 levels.Results Compared with the control group without HG-induction,the protein expression of FTO(0.51±0.04 vs 1.00±0.03)and PRKD2(0.45±0.03 vs 1.01±0.04)was significantly down-regulated in HG-induced podocytes,and the differences were statistically significant(t=13.17,16.76,all P＜0.001).FTO protein levels were positively correlated with PRKD2 protein levels in HG-induced podocytes(r2=0.705 1,P＜0.001).Compared with the vector group,the m6A levels of PRKD2 mRNA(0.56±0.09 vs 1.01±0.13)in the pcDNA-FTO group were decreased,and the mRNA levels of PRKD2(3.16±0.14 vs 1.03±0.02)were increased,with significant differences(t=51.37,11.82,all P＜0.001).Compared with the control group(IL-6:512.76±61.85 pg/ml,TNF-α:28.17±2.83 pg/ml,MCP-1:157.31±17.69 pg/ml)and the vector group(IL-6:498.41±87.51 pg/ml,TNF-α:26.35±5.47 pg/ml,MCP-1:165.52±16.87 pg/ml),the secretion of IL-6(301.86±21.85 pg/ml),TNF-α(11.06±4.12 pg/ml)and MCP-1(81.45±9.03 pg/ml)were significantly decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=7.51,10.47,61.97,all P＜0.01).Compared with the control group(caspase-3 activity:689.65±79.5 U/L,cell apoptosis:22.31%±2.69%)and the vector group(Caspase-3 activity:715.91±113.58 U/L,cell apoptosis:21.07%±3.28%),Caspase-3 activity(437.64±104.76 U/L)and the rate of apoptosis(8.41%±3.15%)were significantly decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=2.35,79.13,all P＜0.01).Compared with the control group(SIRT1:1.01±0.05,HIF-1α:1.03±0.07)and the vector group(SIRT1:0.97±0.05,HIF-1α:1.02±0.03),SIRT1 protein levels(3.51±0.15)were increased and HIF-1α protein levels(0.37±0.07)were decreased in the pcDNA-PRKD2 group,and the differences were statistically significant(F=31.54,8.31,all P＜0.01).Conclusion FTO-mediated and m6A-modified PRKD2 suppresses inflammation and apoptosis in HG-induced podocytes through the SIRT1/HIF-1 pathway.

Protein kinase D2 confers neuroprotection by promoting AKT and CREB activation in ischemic stroke.

Ischemic stroke, constituting 80-90% of all strokes, is a leading cause of death and long-term disability in adults. There is an urgent need to discover new targets and therapies for this devastating condition. Protein kinase D (PKD), as a key target of diacylglycerol involved in ischemic responses, has not been well studied in ischemic stroke, particularly PKD2. In this study, we found that PKD2 expression and activity were significantly upregulated in the ipsilateral side of the brain after transient focal cerebral ischemia, which coincides with the upregulation of PKD2 in primary neurons in response to in vitro ischemia, implying a potential role of PKD2 in neuronal survival in ischemic stroke. Using kinase-dead PKD2 knock-in (PKD2-KI) mice, we examined whether loss of PKD2 activity affected stroke outcomes in mice subjected to 1 h of transient middle cerebral artery occlusion (tMCAO) and 24 h of reperfusion. Our data demonstrated that PKD2-KI mice exhibited larger infarction volumes and worsened neurological scores, indicative of increased brain injury, as compared to the wild-type (WT) mice, confirming a neuroprotective role of PKD2 in ischemia/reperfusion (I/R) injury. Mouse primary neurons obtained from PKD2-KI mice also exhibited increased cell death as compared to the WT neurons when subjected to in vitro ischemia. We have further identified AKT and CREB as two main signaling nodes through which PKD2 regulates neuronal survival during I/R injury. In summary, PKD2 confers neuroprotection in ischemic stroke by promoting AKT and CREB activation and targeted activation of PKD2 may benefit neuronal survival in ischemic stroke.

Pharmacological inhibition of protein kinase D suppresses epithelial ovarian cancer via MAPK/ERK1/2/Runx2 signalling axis.

Epithelial ovarian cancer (EOC) is the most lethal gynaecological malignancy with poor prognosis and dismal patient survival. Although protein kinase D (PKD) isoforms, especially PKD2 and PKD3 are critical for many cellular and physiological functions involved in carcinogenesis including cell proliferation and angiogenesis, their role in human EOC remains unknown. Towards the goal to identify novel prognostic biomarker and therapeutic interventions against EOC, this study aimed to elucidate the molecular roles of PKD2, PKD3 and highly selective, pan-PKD inhibitor CRT0066101 in this lethal pathology. Our results indicated that inactivation of PKD2 and PKD3 by 1 µM CRT0066101 suppressed EOC cell proliferation, colony formation, cell migration and invasion. Moreover, CRT0066101 induced apoptosis and inhibited cell cycle at G2-M phase in EOC cells. Genetic knockdown of PKD2 and PKD3 confirmed the anti-carcinogenic effects of CRT0066101 against EOC. The anti-cancer phenotype of EOC cells resulted from CRT0066101-mediated PKD2 and PKD3 inactivation or genetic depletion was, in part, mediated by transcription factor Runx2 as abrogation of PKD2 and PKD3 caused downregulation of Runx2 and its downstream target genes including osteopontin, focal adhesion kinase and ERK1/2. Moreover, overexpression of a constitutively active PKD2 augmented the expression levels of phosphor-ERK1/2T202/Y204, Runx2 and its downstream targets. Mechanistically, PKD2 and PKD3 positively regulated Runx2 via MAPK/ERK1/2 pathway and promoted EOC. Taken together, our results indicated that PKD2/3/ERK1/2/Runx2 signalling axis might be a novel drug target against EOC and CRT0066101 could be developed as a promising therapeutic choice against this lethal pathology.