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.

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.

Protein Kinase D1 Correlates with Less Lymph Node Metastasis Risk, Enhanced 5-FU Sensitivity, and Better Prognosis in Colorectal Cancer.

Protein kinase D1 (PKD1) controls tumor growth and invasion of gastrointestinal tract-related cancers, but its prognostic role in colorectal cancer (CRC) is not clear yet. Therefore, this research intended to assess the potential of PKD1 as a marker for CRC patients' management, also to evaluate its effect on 5-fluorouracil (5-FU) chemosensitivity in CRC cell lines. PKD1 protein and mRNA expressions were measured by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction assays in 214 CRC patients, respectively. The PKD1 overexpression plasmids and negative control (NC) plasmids were transfected into the HCT-116 and LoVo cell lines followed by 0-16 µM 5-FU treatment. PKD1 protein (P < 0.001) and mRNA expressions (P < 0.001) were both descended in tumor tissues compared to tumor-adjacent tissues. Meanwhile, tumor PKD1 protein and mRNA expressions were both negatively related to lymph node metastasis, N stage, and tumor-node-metastasis (TNM) stage (all P < 0.05). Prognostically, high expressions of PKD1 protein and mRNA were linked with prolonged disease-free survival (DFS) and overall survival (OS) (all P < 0.05). After adjustment by multivariate Cox analyses, PKD1 mRNA high expression independently forecasted longer DFS [hazard ratio (HR) = 0.199, P = 0.002] and OS (HR = 0.212, P = 0.022). In vitro experiments revealed that PKD1 overexpression decreased the half maximal inhibitory concentration value of 5-FU in the HCT-116 (P = 0.016) and LoVo (P = 0.007) cell lines. PKD1 expression links with less lymph node metastasis risk and satisfied prognosis in CRC patients, which promotes CRC cell chemosensitivity to 5-FU chemosensitivity as well.

Protein kinase D1 overexpression potentiates epidermal growth factor signaling pathway in MCF-7 cells.

BACKGROUND: Protein kinase D1, PKD1, is a serine-threonine kinase implicated in cell proliferation, migration, invasion, and/or apoptosis and its activation by several growth factors sets this enzyme as a key regulator of tumorigenesis and tumor progression. Despite many studies, its role in the regulation of intracellular signaling pathways remains widely disparate and needs to be clarified. METHODS AND RESULTS: By using human breast cancer cells MCF-7, overexpressing or not PKD1, we demonstrated that PKD1 expression level modulated the tumor growth-promoting epidermal growth factor (EGF) signaling pathway. We also showed that EGF acutely stimulated PKD1 phosphorylation with similar time courses both in control and PKD1-overexpressing cells. However, PKD1 overexpression specifically and markedly increased EGF-induced phosphorylation of Akt (onto T308 and S473 residues) and extracellular-regulated protein kinase (ERK1/2). Finally, pharmacological inhibition of PKD1 activity or lowering its expression level using specific siRNAs drastically reduced EGF-stimulated Akt and ERK phosphorylation in PKD1overexpressing cells, but not in control cells. CONCLUSIONS: Overall, these results identified the level of PKD1 expression as a key determinant in the regulation of the EGF signaling pathway highlighting its crucial role in a tumorigenic setting.

Osteoblastic protein kinase D1 contributes to the prostate cancer cells dormancy via GAS6-circadian clock signaling.

Disseminated prostate cancer (PCa) is known to have a strong propensity for bone marrow. These disseminated tumor cells (DTCs) can survive in bone marrow for years without obvious proliferation, while maintaining the ability to develop into metastatic lesions. However, how DTCs kept dormant and recur is still uncertain. Here, we focus on the role of osteoblastic protein kinase D1 (PKD1) in PCa (PC-3 and DU145) dormancy using co-culture experiments. Using flow cytometry, western blotting, and immunofluorescence, we observed that in co-cultures osteoblasts could induce a dormant state in PCa cells, which is manifested by a fewer cell divisions, a decrease Ki-67-positive populations and a lower ERK/p38 ratio. In contrast, silencing of PKD1 gene in osteoblasts impedes co-cultured prostate cancer cell's dormancy ability. Mechanismly, protein kinase D1 (PKD1) in osteoblasts induces PCa dormancy via activating CREB1, which promoting the expression and secretion of growth arrest specific 6 (GAS6). Furthermore, GAS6-induced dormancy signaling significantly increased the expression of core circadian clock molecules in PCa cells, and a negative correlation of circadian clock proteins (BMAL1, CLOCK and DEC2) with recurrence-free survival is observed in metastatic prostate cancer patients. Interestingly, the expression of cell cycle factors (p21, p27, CDK1 and PCNA) which regulated by circadian clock also upregulated in response to GAS6 stimulation. Taken together, we provide evidence that osteoblastic PKD1/CREB1/GAS6 signaling regulates cellular dormancy of PCa cells, and highlights the importance of circadian clock in PCa cells dormancy.

Protein kinase D1 induced epithelial-mesenchymal transition and invasion in salivary adenoid cystic carcinoma via E-cadherin/Snail regulation.

Salivary adenoid cystic carcinoma (SACC) is a malignant tumor, which is characterized by a higher incidence of distant metastasis. The aim of this study was to investigate the role and mechanism of protein kinase D1 (PKD1) in regulating the epithelial-mesenchymal transition (EMT) and promotes the metastasis in SACC. We analyzed the expression of PKD1 in 40 SACC patients and different metastatic potential cell lines. Then, we investigated whether the migration and growth of SACC were regulated by PKD1 using shRNA interference or inhibition of kinase active in vitro cell. Moreover, the mechanism by which PKD1 regulates the stability of Snail protein was determined. Finally, nude mice were used to testify the function of PKD1 via tail vein injection. PKD1 was correlated with metastasis and poor prognosis of SACC patients. PKD1 inhibition attenuated proliferation, migration, invasion, and EMT of SACC cells. Conversely, kinase active PKD1 could induce EMT and promoted cell migration in human HSG cell. Furthermore, downregulation of PKD1 regulated Snail via phosphorylation at Ser-11 on Snail protein and promotion of proteasome-mediated degradation, and reduced lung metastasis in vivo. Our results suggest that PKD1 induces the EMT and promotes the metastasis, which illustrate that PKD1 may be a potential prognostic biomarker and serve as a potential therapeutic target for SACC patients.

An Oxidative Stress-Related Gene Pair (CCNB1/PKD1), Competitive Endogenous RNAs, and Immune-Infiltration Patterns Potentially Regulate Intervertebral Disc Degeneration Development.

Oxidative stress (OS) irreversibly affects the pathogenesis of intervertebral disc degeneration (IDD). Certain non-coding RNAs act as competitive endogenous RNAs (ceRNAs) that regulate IDD progression. Analyzing the signatures of oxidative stress-related gene (OSRG) pairs and regulatory ceRNA mechanisms and immune-infiltration patterns associated with IDD may enable researchers to distinguish IDD and reveal the underlying mechanisms. In this study, OSRGs were downloaded and identified using the Gene Expression Omnibus database. Functional-enrichment analysis revealed the involvement of oxidative stress-related pathways and processes, and a ceRNA network was generated. Differentially expressed oxidative stress-related genes (De-OSRGs) were used to construct De-OSRG pairs, which were screened, and candidate De-OSRG pairs were identified. Immune cell-related gene pairs were selected via immune-infiltration analysis. A potential long non-coding RNA-microRNA-mRNA axis was determined, and clinical values were assessed. Eighteen De-OSRGs were identified that were primarily related to intricate signal-transduction pathways, apoptosis-related biological processes, and multiple kinase-related molecular functions. A ceRNA network consisting of 653 long non-coding RNA-microRNA links and 42 mRNA-miRNA links was constructed. Three candidate De-OSRG pairs were screened out from 13 De-OSRG pairs. The abundances of resting memory CD4+ T cells, resting dendritic cells, and CD8+ T cells differed between the control and IDD groups. CD8+ T cell infiltration correlated negatively with cyclin B1 (CCNB1) expression and positively with protein kinase D1 (PKD1) expression. CCNB1-PKD1 was the only pair that was differentially expressed in IDD, was correlated with CD8+ T cells, and displayed better predictive accuracy compared to individual genes. The PKD1-miR-20b-5p-AP000797 and CCNB1-miR-212-3p-AC079834 axes may regulate IDD. Our findings indicate that the OSRG pair CCNB1-PKD1, which regulates oxidative stress during IDD development, is a robust signature for identifying IDD. This OSRG pair and increased infiltration of CD8+ T cells, which play important roles in IDD, were functionally associated. Thus, the OSRG pair CCNB1-PKD1 is promising target for treating IDD.

Adipose-specific knockout of Protein Kinase D1 suppresses de novo lipogenesis in mice via SREBP1c-dependent signaling.

Having healthy adipose tissue is essential for metabolic health, as excessive adipose tissue in the body can cause its dysregulation and driving chronic metabolic diseases. Protein kinase D1 (PKD1) is considered to be a key kinase in signal transduction, which regulates multiple cellular functions, but its physiological functions in adipose are still not fully understood. This study aimed at elucidating the function of adipocyte PKD1 on lipogenesis. From RNA-Sequencing data, we found that the fatty acid biosynthesis pathway in white adipose tissue lacking PKD1 was significantly affected. Critical rate-limiting enzymes for de novo lipogenesis in adipocytes, such as FASN, ACCα, and SCD1, were significantly repressed after deleting PKD1 in vivo and in vitro. Further studies revealed that blockade of PKD1 significantly increased phosphorylation of SREBP1c at serine 372 site. Co-immunoprecipitation analysis showed that PKD1 interacts with SREBP1c in vitro and in vivo. Importantly, overexpression of SREBP1c reversed the inhibition of FASN and ACCα expression caused by PKD1 silencing. Together, adipocyte PKD1 promotes de novo lipogenesis via SREBP1c-dependent manner in visceral white adipose tissue and might provide a new target for the development of anti-obesity therapies.

Higher tumor protein kinase D1 correlates with increased tumor size, BCLC stage, CA199 level, AFP level and worse overall survival in hepatocellular carcinoma patients.

OBJECTIVE: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. Protein kinase D1 (PKD1) is recognized as a key regulator in the progression in several solid cancers, while its clinical role in HCC is unclear. This study aimed to evaluate the correlation of PKD1 with clinical features and prognosis in HCC patients. METHODS: A total of 218 HCC patients who underwent resection were retrospectively enrolled. PKD1 expression in tumor (N = 218) and adjacent (N = 110) tissues was detected by immunohistochemical staining, scored by a semi-quantitative scoring method ranging from 0 to 12, and further classified as PKD1-, PKD1+, PKD1++ and PKD1+++ for analysis. Meanwhile, patients' clinical features and survival data were acquired from the database. RESULTS: PKD1 was elevated in tumor tissues compared with adjacent tissues. Meanwhile, higher tumor PKD1 was correlated with elevated tumor size, Barcelona Clinic Liver Cancer (BCLC) stage, carbohydrate antigen 199 (CA199) level and alpha fetoprotein (AFP) level; while no correlation was found in tumor PKD1 with patients' basic features or liver function indexes. Moreover, higher tumor PKD1 was correlated with worse overall survival (OS) in HCC patients, then further validated as an independent predictive factor for worse OS by multivariate Cox's regression model analysis. Additionally, in Child-Pugh stage A, Child-Pugh stage B, BCLC stage 0/A, and BCLC stage B subgroups, higher tumor PKD1 was also correlated with worse OS. CONCLUSION: Higher PKD1 in tumor tissues correlates with elevated BCLC stage, bigger tumor size, increased CA199 level, higher AFP level and worse OS in HCC patients.

Telangiectasia-ectodermal dysplasia-brachydactyly-cardiac anomaly syndrome is caused by de novo mutations in protein kinase D1.

BACKGROUND: We describe two unrelated patients who display similar clinical features including telangiectasia, ectodermal dysplasia, brachydactyly and congenital heart disease. METHODS: We performed trio whole exome sequencing and functional analysis using in vitro kinase assays with recombinant proteins. RESULTS: We identified two different de novo mutations in protein kinase D1 (PRKD1, NM_002742.2): c.1774G>C, p.(Gly592Arg) and c.1808G>A, p.(Arg603His), one in each patient. PRKD1 (PKD1, HGNC:9407) encodes a kinase that is a member of the protein kinase D (PKD) family of serine/threonine protein kinases involved in diverse cellular processes such as cell differentiation and proliferation and cell migration as well as vesicle transport and angiogenesis. Functional analysis using in vitro kinase assays with recombinant proteins showed that the mutation c.1808G>A, p.(Arg603His) represents a gain-of-function mutation encoding an enzyme with a constitutive, lipid-independent catalytic activity. The mutation c.1774G>C, p.(Gly592Arg) in contrast shows a defect in substrate phosphorylation representing a loss-of-function mutation. CONCLUSION: The present cases represent a syndrome, which associates symptoms from several different organ systems: skin, teeth, bones and heart, caused by heterozygous de novo mutations in PRKD1 and expands the clinical spectrum of PRKD1 mutations, which have hitherto been linked to syndromic congenital heart disease and limb abnormalities.

Protein Kinase D1 Is Increased in Tumor Tissue, Correlates With Advanced Tumor Features and Worse Prognosis of Non-Small Cell Lung Cancer.

OBJECTIVE: This study aimed to assess protein kinase D1 expression and its association with tumor characteristics as well as prognosis in patients with non-small cell lung cancer. METHODS: Protein kinase D1 expression in tumor tissues and adjacent tissues from 172 patients with non-small cell lung cancer who underwent surgical resection were analyzed by immunohistochemical staining. Based on the total immunohistochemical score, protein kinase D1 expression was classified as protein kinase D1 high expression (further divided into protein kinase D1 high+++, protein kinase D1 high++, and protein kinase D1 high+ expressions) and protein kinase D1 low expression. Clinical characteristics of patients with non-small cell lung cancer were acquired from the database. Accumulating disease-free survival and overall survival were calculated based on patients' relapse/survival status. RESULTS: Protein kinase D1 expression was increased in tumor tissues compared to adjacent tissues (P < .001). Tumor protein kinase D1 high expression correlated with poorer pathological differentiation (P = .041), increased tumor size (P = .003), the presence of lymph node metastasis (P = .001), and elevated tumor, nodes and metastases stage (P < .001). Besides, both accumulating disease-free survival and overall survival were decreased in patients with tumor protein kinase D1 high expression compared to patients with tumor protein kinase D1 low expression (P = .010 for disease-free survival and P = 0.005 for overall survival). Moreover, they were lowest in patients with tumor protein kinase D1 high+++ expression, followed by patients with tumor protein kinase D1 high++ expression, then patients with tumor protein kinase D1 high+ expression, and highest in patients with tumor protein kinase D1 low expression (P < .001 for disease-free survival and P = .001 for overall survival). Notably, higher tumor protein kinase D1 expression was an independent predictive factor for decreased disease-free survival (P = .001) and overall survival (P = .004). CONCLUSIONS: Protein kinase D1 might be a potential marker to identify patients with non-small cell lung cancer with worse tumor features and prognosis.

Protein Kinase D 1 Predicts Poor Treatment Response and Unfavorable Survival of Bortezomib-Based Treatment, and Its Knockdown Enhances Drug Sensitivity to Bortezomib in Multiple Myeloma.

OBJECTIVE: The present study aimed to explore the correlation of protein kinase D 1 with prognosis in bortezomib-treated multiple myeloma patients and further investigate the effect of protein kinase D 1 knockdown on drug sensitivity to bortezomib in multiple myeloma cells. METHODS: Totally, 104 de novo symptomatic multiple myeloma patients treated with bortezomib-based regimens and 30 healthy controls were recruited. Bone marrow mononuclear cells-derived plasma cells were collected from multiple myeloma patients before initial treatment and from healthy controls on the bone marrow donation, respectively, then protein kinase D 1 protein/messenger RNA expressions were detected by Western blot and reverse transcription quantitative polymerase chain reaction, respectively. The effect of protein kinase D 1 knockdown on drug sensitivity to bortezomib was detected by transfecting protein kinase D 1 knockdown plasmid and control plasmid into RPMI8226 and U266 cells. RESULTS: Protein kinase D 1 protein/messenger RNA expressions were both upregulated in multiple myeloma patients compared with healthy controls and presented good value in differentiating multiple myeloma patients from healthy controls. Furthermore, protein kinase D 1 protein/messenger RNA expressions were both associated with high International Staging System stage and t (4; 14). Furthermore, both complete response rate and overall response rate were reduced in protein kinase D 1 high patients compared with protein kinase D 1 low patients; similarly, progression-free survival and overall survival were both decreased in protein kinase D 1 high patients compared with protein kinase D 1 low patients. In addition, in RPMI8226 and U266 multiple myeloma cells, protein kinase D 1 knockdown increased drug sensitivity to bortezomib. CONCLUSION: Protein kinase D 1 has the potential to predict poor treatment response and unfavorable survival of bortezomib-based treatment in multiple myeloma patients, and its knockdown enhanced drug sensitivity to bortezomib in multiple myeloma cells.

FAM83G Is a Novel Inducer of Apoptosis.

The family with sequence similarity 83 (FAM83) protein family G (FAM83G) possesses a predicted consensus phosphorylation motif for serine/threonine-protein kinase D1/protein kinase C mu (PKD1/PKCµ) at serine residue 356 (S356). In this study, overexpressed wild-type FAM83G coimmunoprecipitated with PKD1/PKCµ in Chinese hamster ovary (CHO) cells inhibited heat shock protein 27 (HSP27) phosphorylation at S82 and reduced the living cell number. The expression of a FAM83G phosphorylation-resistant mutant (S356A-FAM83G) had no effect on the living cell number or the induction of spontaneous apoptosis. By contrast, the introduction of a synthetic peptide encompassing FAM83G S356 into HCT116 and HepG2 cells decreased HSP27 S15 and S82 phosphorylation and induced spontaneous apoptosis. On the other hand, the introduction of FAM83G phosphorylation-resistant mutant synthesized peptides (S356A-AF-956 and S356A-AG-066) did not reduce the living cell number or induce spontaneous apoptosis. The endogenous expression of HSP27 and FAM83G was apparently greater in HCT116 and HepG2 cells compared with in CHO cells. In various types of lung cancer cell lines, the FAM83G messenger RNA (mRNA) level in non-small lung cancer cells was at a similar level to that in non-cancerous cells. However, the FAM83G mRNA level in the small cell lung cancer cell lines was variable, and the HSP27 mRNA level in FAM83G mRNA-rich types was greater than that in FAM83G mRNA-normal range types. Taken together, these data demonstrate that FAM83G S356 phosphorylation modulates HSP27 phosphorylation and apoptosis regulation and that HSP27 is a counterpart of FAM83G.

The L1 cell adhesion molecule affects protein kinase D1 activity in the cerebral cortex in a mouse model of Alzheimer's disease.

Alzheimer's disease (AD) is characterized by deposition of ß-amyloid protein (Aß), neurofibrillary tangles and cognitive deficits resulting from neuronal cell death. In search for the molecular underpinnings of the disease, we were interested in the relationship between Aß, L1 cell adhesion molecule and protein kinase D1 (PKD1), which are not only implicated in neural development and functional maintenance in the adult, but are also neuroprotective under pathological conditions. Based on our observations that L1 and phosphorylated, i.e. activated, protein kinase PKD1 (pPKD1) co-localize in cultured neurons, we investigated the functional relationship between L1 and pPKD1 in the frontal lobe of an AD human cortical tissue microarray, and found increased and positively correlating levels of both molecules when compared to a non-affected human brain. Also in the APPSWE mouse model of AD, L1 and pPKD1 levels were increased in the frontal lobe. To investigate whether L1 influences PKD1-based functions in AD, cultured cortical neurons were stressed with either H2O2 or oligomeric Aß1-42, in the presence or absence of recombinant L1 extracellular domain, and PKD1 phosphorylation was measured. As indicated by the cell viability assay, L1 maintained neuronal survival under oxidative stress and under application of oligomeric Aß1-42, when PKD1 activity was inhibited, suggesting that L1 ameliorates some aspects of Aß1-42 pathology in parallel with reducing PKD1 function.

Effect of Danggui Buxuetang on PKD1/NF-κB/MnSOD Signal Pathway in Bleomycin-induced Pulmonary Fibrosis in Rats / 中国实验方剂学杂志

Objective:To observe the effect of Danggui Buxuetang on lung histopathology and protein kinase D1 (PKD1), nuclear transcription factor-κB (NF-κB) and manganese superoxide dismutase (MnSOD)-mediated oxidative stress pathway in rats with pulmonary fibrosis induced by bleomycin, so as to explore the mechanism of intervention of pulmonary fibrosis.Method:Thirty-two male SPF SD rats were randomly divided into sham operation group, model group, Danggui Buxuetang group and prednisone group, with 8 rats in each group. Except the sham operation group, the other groups were prepared through the intratracheal instillation with bleomycin. After modeling for 24 h, the rats of Danggui Buxuetang group were administered with Danggui Buxuetang (0.81 g·kg-1). The rats of prednisone group were given aqueous solution of prednisone (0.005 g·kg-1). The rats of sham operation group and model group were given the same volume of saline. After 14 days of administration, blood was collected from the femoral artery, serum was separated, and the lungs were taken by thoracotomy. The pathological changes of rat lung tissues were observed by hematoxylin-eosin staining (HE) and Masson trichrome staining, and graded by Szapiel score and Ashcroft score at the same time. The content of serum malondialdehyde (MDA), and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) were determined. Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR) and Western blot were used to measure mRNA and protein expressions of PKD1, NF-κB, MnSOD.Result:Compared with the rats in sham operation group, the rats in model group had higher Szapiel scores and Ashcroft scores (P<0.05), higher serum MDA content , but lower SOD, CAT and GSH-Px activities(P<0.01), moreover, the rat lung tissues in model group had higher mRNA and protein expressions of PKD1, NF-κB and MnSOD (P<0.01) than those in sham operation group. Compared with the rats in model group, the Szapiel scores and Ashcroft scores of the rats in Danggui Buxuetang group were decreased significantly（P<0.05）. The serum MDA content was decreased significantly, and SOD, CAT, GSH-Px activities were increased, whereas mRNA and protein expressions of PKD1, NF-κB, MnSOD in the rat lung tissues were decreased（P<0.05，P<0.01）.Conclusion:Danggui Buxuetang can reduce the degree of pulmonary fibrosis by regulating the anti-oxidation pathway of PKD1/NF-κB/MnSOD mitochondrial nucleus and improving the body's antioxidant capacity.

[Protein kinase D1 regulates the growth and metabolism of oral squamous carcinoma cells in tumor microenvironment].

OBJECTIVE: To observe the effect of protein kinase D1 (PKD1) on the growth and metabolism of oral squamous cell carcinoma HSC-4 cells and related molecular mechanisms in the tumor microenvironment. METHODS: HSC-4 cell lines were transfected with shRNA plasmids. Three groups (Wild, control-shRNA, and PKD1-shRNA) were cultured under acidic or hypoxic environment for a certain time. Western blot was used to detect the expression of autophagy-related and glycolytic-related proteins. The proliferation changes were detected by CCK-8 kits. RESULTS: The PKD1-knockdown HSC-4 cell line was established. PKD1 silencing increased autophagy activity. Under hypoxic and acidic conditions, the PKD1-knockdown HSC-4 cells showed lower proliferation than the parental cells. PKD1-knockdown also decreased the expression of hypoxia induciblefactor 1α (HIF-1α) and pyruvate kinase M2 (PKM2). CONCLUSIONS: Under hypoxic and acidic conditions, PKD1 gene silencing can increase apoptotic autophagy activity. Downregulated PKD1 gene expression can reduce the glycolysis of oral squamous cell carcinoma cells and inhibit tumor cell proliferation. This study revealed the important role of PKD1 in the metabolism and growth of oral squamous cell carcinoma, making it a possible target for the treatment of oral squamous cell carcinoma.

PLD1 promotes dendritic spine morphogenesis via activating PKD1.

Dendritic spines on the dendrites of pyramidal neurons are one of the most important components for excitatory synapses, where excitatory information exchanges and integrates. The defects of dendritic spine development have been closely connected with many nervous system diseases including autism, intellectual disability and so forth. Based on our previous studies, we here report a new functional signaling link between phospholipase D1 (PLD1) and protein kinase D1 (PKD1) in dendritic spine morphogenesis. Coimmunoprecipitation assays showed that PLD1 associates with PKD1. A series of knocking down and rescuing experiments demonstrated that PLD1 acts upstream of PKD1 in positively regulating dendritic spine morphogenesis. Using PLD1 inhibitor, we found that PLD1 activates PKD1 to promote dendritic spine morphogenesis. Thus, we further reveal the roles of the two different enzymes in neuronal development.

Protein kinase D1 induces G1-phase cell-cycle arrest independent of Checkpoint kinases by phosphorylating Cell Division Cycle Phosphatase 25.

Protein Kinase D1 (PrKD1) functions as a tumor and metastasis suppressor in several human cancers by influencing cell-cycle progression. However, the exact mechanism of cell-cycle regulation by PrKD1 is unclear. Overexpression and ectopic expression of PrKD1 induces G1 arrest in cancer cell lines. Because checkpoint kinases (CHEKs) are known to play a role in progression through the G1 phase, we downregulated CHEK1, which did not overcome the G1 arrest induced by PrKD1. Using in vitro phosphorylation and Western blot assays, we showed that PrKD1 phosphorylates all CDC25 isoforms (known substrates of CHEK kinases), independent from CHEK kinases, suggesting that direct phosphorylation of CDC25 by PrKD1 may be an alternate mechanism of G1 arrest. The study has identified a molecular mechanism for the influence of PrKD1 in cell-cycle progression.

Angiogenic function of astragaloside IV in rats with myocardial infarction occurs via the PKD1-HDAC5-VEGF pathway.

The current study aimed to assess the role and mechanism of astragaloside IV (AS-IV) in myocardial infarction. A myocardial infarction model was established via the ligation of the left anterior descending artery. Rats were randomly divided into sham, DMSO, model, AS-IV, AS-IV-CID755673 and CID755673 inhibitor groups. Rats were then sacrificed following 4 weeks of treatment and segmental heart samples were obtained for hematoxylin and eosin, and masson staining. The expression of PKD1, HDAC5 and VEGF were analyzed using immunohistochemistry, reverse transcription polymerase chain reaction and western blotting. Compared with the sham and DMSO groups, the morphology of myocardium in the model and CID755673 inhibitor groups were disordered and exhibited necrotic myocardial cells and collagen tissues. Following treatment with AS-IV, the morphology of the myocardium was markedly improved and the number of new blood vessels increased. However, following treatment with CID755673, the myocardial tissue of rats became disordered, with an increased number of necrotic cells and the closure of certain vessels. The expression of PKD1, HDAC5 and VEGF mRNA and protein in myocardial tissue of model group and CID755673 inhibitor group were significantly lower than the other four groups (P<0.05), whereas these levels in the AS-IV group were significantly higher than those in the other five groups (P<0.01). Additionally, the AS-IV-CID755673 group exhibited significantly higher levels of PKD1, HDAC5 and VEGF mRNA and protein than the sham, DMSO, CID755673 inhibitor and model groups (P<0.05). Furthermore, the protein expression of pS205 PKD1, pS259 HDAC5 and pTyr951 VEGF in the myocardium of rats was comparable with that of PKD1, HDAC5 and VEGF. AS-IV may partly promote the angiogenesis of myocardial tissue in rats with myocardial infarction via the PKD1-HDAC5-VEGF pathway.