Protein Kinase D Activation Research Articles

Protein kinase D1 in myeloid lineage cells contributes to the accumulation of CXCR3+CCR6+ nonconventional Th1 cells in the lungs and potentiates hypersensitivity pneumonitis caused by S. rectivirgula.

Hypersensitivity pneumonitis (HP) is an extrinsic allergic alveolitis characterized by inflammation of the interstitium, bronchioles, and alveoli of the lung that leads to granuloma formation. We previously found that activation of protein kinase D1 (PKD1) in the lungs following exposures to Saccharopolyspora rectivirgula contributes to the acute pulmonary inflammation, IL-17A expression in the lungs, and development of HP. In the present study, we investigated whether PKD1 in myeloid-lineage cells affects the pathogenic course of the S. rectivirgula-induced HP. Mice were exposed intranasally to S. rectivirgula once or 3 times/week for 3 weeks. The protein and mRNA expression levels of cytokines/chemokines were detected by enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. Flow cytometry was used to detect the different types of immune cells and the levels of surface proteins. Lung tissue sections were stained with hematoxylin and eosin, digital images were captured, and immune cells influx into the interstitial lung tissue were detected. Compared to control PKD1-sufficient mice, mice with PKD1 deficiency in myeloid-lineage cells (PKD1mKO) showed significantly suppressed expression of TNFα, IFNγ, IL-6, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10 and neutrophilic alveolitis after single intranasal exposure to S. rectivirgula. Substantially reduced levels of alveolitis and granuloma formation were observed in the PKD1mKO mice repeatedly exposed to S. rectivirgula for 3 weeks. In addition, expression levels of the Th1/Th17 polarizing cytokines and chemokines such as IFNγ, IL-17A, CXCL9, CXCL10, CXCL11, and CCL20 in lungs were significantly reduced in the PKD1mKO mice repeatedly exposed to S. rectivirgula. Moreover, accumulation of CXCR3+CCR6+ nonconventional Th1 in the lungs were significantly reduced in PKD1mKO mice repeatedly exposed to S. rectivirgula. Our results demonstrate that PKD1 in myeloid-lineage cells plays an essential role in the development and progress of HP caused by repeated exposure to S. rectivirgula by contributing Th1/Th17 polarizing proinflammatory responses, alveolitis, and accumulation of pathogenic nonconventional Th1 cells in the lungs.

Abstract P2039: Alteration In Intercalated Disc Composition Impacts Cardiac Disease Progression

Intercalated discs (IDs) connect cardiomyocytes electro-mechanically and ID dysfunction has been associated with the onset of cardiomyopathies and arrhythmias. The ID- and myofibril-associated protein nebulin-related anchoring protein (NRAP) was identified as a putative novel interaction partner of protein kinase D1 (PKD). PKD1 phosphorylates class IIa histone deacetylases (HDAC), leading to their cytosolic export. This causes a de-repression of HDAC-mediated inhibition of myocyte enhancer factor 2 (MEF2)-dependent fetal and pro-hypertrophic gene expression. We investigated the regulation of NRAP by PKD1 in the developing and diseased heart. PKD activity was modulated by using the pharmacological PKD inhibitor BPKDi and/or the pro-hypertrophic stimulus phenylephrine (PE) in neonatal rat ventricular myocytes (NRVMs) and -derived engineered heart tissues (EHTs). NRVMs were analyzed by immunofluorescence, western blotting and stable isotope labelling with amino acids in cell culture/mass spectrometry. The outcome was studied by exposure to an autophagy (bafilomycin A1) or a proteasomal (MG132) inhibitor or performing proteome analysis from hearts of MuRF-1/3 double-knock-out (DKO) mice. Exposure of NRVMs to PE enhanced myofibril maturation and NRAP translocation towards IDs while BPKDi led to perinuclear accumulation of NRAP and improved force in EHTs. Combined BPKDi and PE disturbed sarcomeric structure and prevented NRAP localization at the ID. Proteomic analysis revealed that BPKDi reduced NRAP abundance and enhanced the abundance of proteins involved in proteasomal, lysosomal and spliceosomal pathways. Inhibition of proteasomal protein degradation by MG132 increased NRAP levels, whereas NRAP was 42-fold more abundant in hearts of MuRF-1/3-DKO mice that show a protein surplus cardiomyopathy. In conclusion, PKD1 impacts NRAP expression and translocation, and negatively affects EHT force development while proteasomal inhibition increased NRAP abundance suggesting a regulation via the PKD1-HDAC-MEF2 axis. Further investigations aim to study pathways involving PKD1 to regulate myofibril assembly and maintenance in the development and progression of cardiac disease.

Deletion of protein kinase D1 in myeloid lineage cells suppresses development of IFNγ +IL-17A +CD4 T cells and ameliorates hypersensitivity pneumonitis

Abstract Hypersensitivity pneumonitis (HP) is an extrinsic allergic alveolitis characterized by inflammation of the interstitium, bronchioles, and alveoli of the lung that leads to granuloma formation and ultimately irreversible fibrosis. Previous studies found that exposure to HP inciting antigen S. rectivirgula(SR) leads to the activation of protein kinase D1 (PKD1) in the mouse lung in vivoand that PKD1 contributes to the acute pulmonary inflammation, IL-17A expression in the lungs, and development of HP caused by repeated exposure to SR. In the present study, we investigated whether PKD1 in myeloid cells affects the pathogenic course of the SR-induced HP. Compared to control PKD1-sufficient mice, mice with deficiency of PKD1 in myeloid cells (PKD1mKO) showed significantly suppressed expression of IL-6, TNFα, IFNγ, CCL2, CCL3, CCL4, CXCL1, CXCL2, and CXCL10 and neutrophilic alveolitis after single intranasal exposure to SR. Substantially reduced levels of alveolitis and granuloma formation were observed in the PKD1mKO mice repeatedly exposed to SR. Surface expression of MHC-II and CD86 on myeloid cells, and expression of IFNγ, IL-17A, CCL20, CXCL9, and CXCL10 in lungs were significantly reduced in the PKD1mKO mice repeatedly exposed to SR. In addition, surface expression of CD69, CXCR3, and CCR6 on CD4 T cells and frequency of IFNγ +IL-17A +CD4 T cells in the lungs were significantly reduced in PKD1mKO mice repeatedly exposed to SR. Our results demonstrate PKD1 in myeloid lineage cells plays an essential role in the initial proinflammatory responses and neutrophil influx in the lung after exposed to SR and contributes to the development of IFNγ +IL-17A +CD4 T cells and HP caused by repeated exposure to SR. Supported by grants from NIH (R01 AR064723, R01 AR069010) and UTHSC.

Endothelial Protein kinase D1 is a major regulator of post-traumatic hyperinflammation.

Trauma is a major cause of death worldwide. The post-traumatic immune response culminates in the release of pro-inflammatory mediators, translating in the infiltration of neutrophils (PMNs) at injury sites. The extent of this inflammation is determined by multiple factors, such as PMN adhesion to the endothelium, transendothelial migration, endothelial barrier integrity as well as PMN swarming, mass infiltration and activation. This process is initiated by secondary lipid mediators, such as leukotriene B4 (LTB4). We here provide evidence that Protein kinase D1 (PRKD1) in endothelial cells is implicated in all these processes. Endothelial PRKD1 is activated by pro-inflammatory stimuli and amplifies PMN-mediated inflammation by upregulation of cytokine and chemokines as well as adhesion molecules, such as ICAM-1, VCAM-1 and E-selectin. This induces enhanced PMN adhesion and trans-migration. PRKD1 activation also destabilizes endothelial VE-cadherin adhesion complexes and thus the endothelial barrier, fostering PMN infiltration. We even describe a yet unrecognized PRKD1-dependant mechanism to induce biosynthesis of the PMN-swarming mediator LTB4 directed via intercellular communication through small extracellular vesicles (sEVs) and enhanced CXCL8 secretion from activated endothelial cells. These endothelial sEVs transfer the LTB4 biosynthesis enzyme LTA4 hydrolase (LTA4H) to prime PMNs, while initiating biosynthesis also requires additional signals, like CXCL8. We further demonstrate the respective LTA4H-positive sEVs in the serum of polytrauma patients, peaking 12h post injury. Therefore, PRKD1 is a key regulator in the coordinated communication of the endothelium with PMNs and a vital signaling node during post-traumatic inflammation.

Statins inhibit protein kinase D (PKD) activation in intestinal cells and prevent PKD1-induced growth of murine enteroids.

We examined the impact of statins on protein kinase D (PKD) activation by G protein-coupled receptor (GPCR) agonists. Treatment of intestinal IEC-18 cells with cerivastatin inhibited PKD autophosphorylation at Ser916 induced by angiotensin II (ANG II) or vasopressin in a dose-dependent manner with half-maximal inhibition at 0.2 µM. Cerivastatin treatment inhibited PKD activation stimulated by these agonists for different times (5-60 min) and blunted HDAC5 phosphorylation, a substrate of PKD. Other lipophilic statins, including simvastatin, atorvastatin, and fluvastatin also prevented PKD activation in a dose-dependent manner. Using IEC-18 cell lines expressing PKD1 tagged with EGFP (enhanced green fluorescent protein), cerivastatin or simvastatin blocked GPCR-mediated PKD1-EGFP translocation to the plasma membrane and its subsequent nuclear accumulation. Similar results were obtained in IEC-18 cells expressing PKD3-EGFP. Mechanistically, statins inhibited agonist-dependent PKD activation rather than acting directly on PKD catalytic activity since exposure to cerivastatin or simvastatin did not impair PKD autophosphorylation or PKD1-EGFP membrane translocation in response to phorbol dibutyrate, which bypasses GPCRs and directly stimulates PKC and PKD. Furthermore, cerivastatin did not inhibit recombinant PKD activity determined via an in vitro kinase assay. Using enteroids generated from intestinal crypt-derived epithelial cells from PKD1 transgenic mice as a model of intestinal regeneration, we show that statins oppose PKD1-mediated increase in enteroid area, complexity (number of crypt-like buds), and DNA synthesis. Our results revealed a previously unappreciated inhibitory effect of statins on receptor-mediated PKD activation and in opposing the growth-promoting effects of PKD1 on intestinal epithelial cells.

Pkd1 is necessary for full cardiac AngII-induced hypertrophy but not for full acute AngII-induced calcium handling changes.

The renin-angiotensin system (RAS) regulates blood pressure, extracellular fluid and electrolyte balance, cardiac inotropy, and vascular resistance. In the heart, angiotensin II (AngII) can induce hypertrophy directly by activating Protein Kinase D (PKD), a signaling kinase known to contribute to cardiac remodeling. Little is known, however, about the acute and chronic functional effects of AngII-induced PKD activation on calcium (Ca) handling. To investigate this, we used hearts from cardiac-specific PKD1 knockout (cKO) mice and their WT littermates. We measured Ca transients (CaT) and Ca spark (CaSp) frequencies in cardiomyocytes at 0.5 and 1 Hz pacing under acute and chronic AngII stimulation (100 nM in vitro and 3 mg/kg/day in osmotic minipump, respectively). Sarcoplasmic reticulum (SR) Ca load was assessed by rapid caffeine-induced Ca release. At baseline and after acute AngII treatment, CaT amplitude and tau decay, CaSp frequency and SR Ca load were similar in WT vs. PKD1 cKO. This was also the case for chronic exposure to AngII except that the tau decay was significantly slower in PKD1 cKO myocytes. In agreement with previous reports, echocardiographic measurements revealed that PKD1 cKO mice were also more resistant to chronic AngII-induced hypertrophy (LVPW increase 24.69 ± 2.74% vs. 54.73 ± 7.73 %) and ejection fraction reduction (4.08 ± 1.5% vs. 12.67 ± 0.52%). We conclude that despite PKD being necessary for the remodeling changes induced by chronic AngII, it does not alter the amplitude and kinetics of CaT and spontaneous Ca release, except for the slowed decay kinetics in the PKD1 cKO after chronic AngII treatment and decreased acute SR Ca load.

Protein kinase D: A therapeutic target in experimental alcoholic pancreatitis

BackgroundAlcohol abuse, a main cause of pancreatitis, has been known to augment NF-κB activation and cell necrosis in pancreatitis. However, the underlying mechanisms are unclear. We recently reported that inhibition of protein kinase D (PKD) alleviated NF-κB activation and severity of experimental pancreatitis. Here we investigated whether PKD signaling mediated the modulatory effects of alcohol abuse on pathological responses in alcoholic pancreatitis. MethodsAlcoholic pancreatitis was provoked in two rodent models with pair-feeding control and ethanol-containing Lieber-DeCarli diets for up to 8 weeks followed by up to 7 hourly intraperitoneal injections of cerulein at 1 μg/kg (rats) or 3 μg/kg (mice). Effects of PKD inhibition by PKD inhibitors or genetic deletion of pancreatic PKD isoform (PKD3Δpanc mice) on alcoholic pancreatitis parameters were determined. ResultsEthanol administration amplified PKD signaling by promoting expression and activation of pancreatic PKD, resulted in augmented/promoted pancreatitis responses. Pharmacological inhibition of PKD or with PKD3Δpanc mice prevented the augmenting/sensitizing effect of ethanol on NF-κB activation and inflammatory responses, cell necrotic death and the severity of disease in alcoholic pancreatitis. PKD inhibition prevented alcohol-enhanced trypsinogen activation, mRNA expression of multiple inflammatory molecules, the receptor-interacting protein kinase activation, ATP depletion, and downregulation of pro-survival Bcl-2 protein in alcoholic pancreatitis. Furthermore, PKD inhibitor CID755673 or CRT0066101, administrated after the induction of pancreatitis in mouse and rat alcoholic pancreatitis models, significantly mitigated the severity of pancreatitis. ConclusionPKD mediates effect of alcohol abuse on pathological process of pancreatitis and constitutes a novel therapeutic target to treat this disease.

Evaluation of protein kinase D auto-phosphorylation as biomarker for NLRP3 inflammasome activation.

BackgroundThe NLRP3 inflammasome is a critical component of sterile inflammation, which is involved in many diseases. However, there is currently no known proximal biomarker for measuring NLRP3 activation in pathological conditions. Protein kinase D (PKD) has emerged as an important NLRP3 kinase that catalyzes the release of a phosphorylated NLRP3 species that is competent for inflammasome complex assembly.MethodsTo explore the potential for PKD activation to serve as a selective biomarker of the NLRP3 pathway, we tested various stimulatory conditions in THP-1 and U937 cell lines, probing the inflammasome space beyond NLRP3. We analyzed the correlation between PKD activation (monitored by its auto-phosphorylation) and functional inflammasome readouts.ResultsPKD activation/auto-phosphorylation always preceded cleavage of caspase-1 and gasdermin D, and treatment with the PKD inhibitor CRT0066101 could block NLRP3 inflammasome assembly and interleukin-1β production. Conversely, blocking NLRP3 either genetically or using the MCC950 inhibitor prevented PKD auto-phosphorylation, indicating a bidirectional functional crosstalk between NLRP3 and PKD. Further assessments of the pyrin and NLRC4 pathways, however, revealed that PKD auto-phosphorylation can be triggered by a broad range of stimuli unrelated to NLRP3 inflammasome assembly.ConclusionAlthough PKD and NLRP3 become functionally interconnected during NLRP3 activation, the promiscuous reactivity of PKD challenges its potential use for tracing the NLRP3 inflammasome pathway.

Protein kinase D promotes activity-dependent AMPA receptor endocytosis in hippocampal neurons.

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors (AMPARs) mediate the majority of fast excitatory neurotransmission in the brain. The continuous trafficking of AMPARs into and out of synapses is a core feature of synaptic plasticity, which is considered as the cellular basis of learning and memory. The molecular mechanisms underlying the postsynaptic AMPAR trafficking, however, are still not fully understood. In this work, we demonstrate that the protein kinase D (PKD) family promotes basal and activity-induced AMPAR endocytosis in primary hippocampal neurons. Pharmacological inhibition of PKD increased synaptic levels of GluA1-containing AMPARs, slowed down their endocytic trafficking and increased neuronal network activity. By contrast, ectopic expression of constitutive active PKD decreased the synaptic level of AMPARs, while increasing their colocalization with early endosomes. Our results thus establish an important role for PKD in the regulation of postsynaptic AMPAR trafficking during synaptic plasticity.

Decoding the Cardiac Actions of Protein Kinase D Isoforms.

Protein kinase D (PKD) consists of a family of three structurally related enzymes that play key roles in a wide range of biological functions that contribute to the evolution of cardiac hypertrophy and heart failure. PKD1 (the founding member of this enzyme family) has been implicated in the phosphorylation of substrates that regulate cardiac hypertrophy, contraction, and susceptibility to ischemia/reperfusion injury, and de novo PRKD1 (protein kinase D1 gene) mutations have been identified in patients with syndromic congenital heart disease. However, cardiomyocytes coexpress all three PKDs. Although stimulus-specific activation patterns for PKD1, PKD2, and PKD3 have been identified in cardiomyocytes, progress toward identifying PKD isoform-specific functions in the heart have been hampered by significant gaps in our understanding of the molecular mechanisms that regulate PKD activity. This review incorporates recent conceptual breakthroughs in our understanding of various alternative mechanisms for PKD activation, with an emphasis on recent evidence that PKDs activate certain effector responses as dimers, to consider the role of PKD isoforms in signaling pathways that drive cardiac hypertrophy and ischemia/reperfusion injury. The focus is on whether the recently identified activation mechanisms that enhance the signaling repertoire of PKD family enzymes provide novel therapeutic strategies to target PKD enzymes and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling. SIGNIFICANCE STATEMENT: PKD isoforms regulate a large number of fundamental biological processes, but the understanding of the biological actions of individual PKDs (based upon studies using adenoviral overexpression or gene-silencing methods) remains incomplete. This review focuses on dimerization, a recently identified mechanism for PKD activation, and the notion that this mechanism provides a strategy to develop novel PKD-targeted pharmaceuticals that restrict proliferation, invasion, or angiogenesis in cancer and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling.

LPA1-mediated PKD2 activation promotes LPA-induced tissue factor expression via the p38α and JNK2 MAPK pathways in smooth muscle cells

Tissue factor (TF) is the principal initiator of blood coagulation and is necessary for thrombosis. We previously reported that lysophosphatidic acid (LPA), a potent bioactive lipid, highly induces TF expression at the transcriptional level in vascular smooth muscle cells. To date, however, the specific role of the LPA receptor is unknown, and the intracellular signaling pathways that lead to LPA induction of TF have been largely undetermined. In the current study, we found that LPA markedly induced protein kinase D (PKD) activation in mouse aortic smooth muscle cells (MASMCs). Small-interfering RNA-mediated knockdown of PKD2 blocked LPA-induced TF expression and activity, indicating that PKD2 is the key intracellular mediator of LPA signaling leading to the expression and cell surface activity of TF. Furthermore, our data reveal a novel finding that PKD2 mediates LPA-induced TF expression via the p38α and JNK2 MAPK signaling pathways, which are accompanied by the PKD-independent MEK1/2-ERK-JNK pathway. To identify the LPA receptor(s) responsible for LPA-induced TF expression, we isolated MASMCs from LPA receptor-knockout mice. Our results demonstrated that SMCs isolated from LPA receptor 1 (LPA1)-deficient mice completely lost responsiveness to LPA stimulation, which mediates induction of TF expression and activation of PKD and p38/JNK MAPK, indicating that LPA1 is responsible for PKD2-mediated activation of JNK2 and p38α. Taken together, our data reveal a new signaling mechanism in which the LPA1-PKD2 axis mediates LPA-induced TF expression via the p38α and JNK2 pathways. This finding provides new insights into LPA signaling, the PKD2 pathway, and the mechanisms of coagulation/atherothrombosis.

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.

Dysfunctional EGFR and oxidative stress-induced PKD1 signaling drive formation of DCLK1+ pancreatic stem cells

SummaryDoublecortin-like kinase 1 (DCLK1)-positive pancreatic cancer stem cells develop at a precancerous stage and may contribute to the lack of efficacy of pancreatic cancer therapy. Although PanIN cells express oncogenic KRas and have an increased activity of epidermal growth factor receptor (EGFR), we demonstrate that, in DCLK1+ PanIN cells, EGFR signaling is not propagated to the nucleus. Mimicking blockage of EGFR with erlotinib in PanIN organoid culture or in p48cre;KrasG12D mice led to a significant increase in DCLK1+ PanIN cells. As a mechanism of how EGFR inhibition leads to formation of DCLK1+ cells, we identify an increase in hydrogen peroxide contributing to activation of Protein Kinase D1 (PKD1). Active PKD1 then drives stemness and abundance of DCLK1+ cells in lesions. Our data suggest a signaling mechanism that leads to the development of DCLK1+ pancreatic cancer stem cells, which can be exploited to target this population in potential therapeutic approaches.

The role of protein kinase D (PKD) in intracellular nutrient sensing and regulation of adaptive responses to the obese environment.

Obesity is associated with ectopic accumulation of lipids, which is implicated in the development of insulin resistance, type 2 diabetes mellitus and cardiovascular disease. As the global prevalence of obesity continues to rise, it is becoming increasingly important to understand the underlying cellular mechanisms of this disease. Protein kinase D (PKD) is an intracellular signalling kinase with well characterized roles in intracellular vesicle transport and secretion, cancer cell proliferation and cardiac hypertrophy. However, emerging evidence also highlights PKD as a novel nutrient sensor. PKD activation is mediated by the accumulation of the lipid intermediate diacylglycerol, and PKD activity in the liver, heart and adipose tissue increases upon feeding. In obesity, PKD signalling is linked to reduced insulin signalling and dysfunction in adipose tissue, liver and heart, whilst in the pancreas, PKD is essential for the compensatory increase in glucose-stimulated insulin secretion from β-cells during obesity. Collectively, these studies reveal aspects of PKD signalling that are involved in the tissue-specific responses to obesity. This review summarizes the emerging evidence suggesting that PKD plays an important role in regulating the adaptive response to the obese environment.

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 β-amyloid protein (Aβ), 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 Aβ, 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 Aβ1−42, 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 Aβ1−42, when PKD1 activity was inhibited, suggesting that L1 ameliorates some aspects of Aβ1−42 pathology in parallel with reducing PKD1 function.

Protein Kinase D1, Reduced in Human Pancreatic Tumors, Increases Secretion of Small Extracellular Vesicles From Cancer Cells That Promote Metastasis to Lung in Mice

Pancreatic tumor cells release small extracellular vesicles (sEVs, exosomes) that contain lipids and proteins, RNA, and DNA molecules that might promote formation of metastases. It is not clear what cargo these vesicles contain and how they are released. Protein kinase D1 (PRKD1) inhibits cell motility and is believed to be dysregulated in pancreatic ductal adenocarcinomas. We investigated whether it regulates production of sEVs in pancreatic cancer cells and their ability to form premetastatic niches for pancreatic cancer cells in mice. We analyzed data from UALCAN and human pancreatic tissue microarrays to compare levels of PRKD1 between tumor and nontumor tissues. We studied mice with pancreas-specific disruption of Prkd1 (PRKD1KO mice), mice that express oncogenic KRAS (KC mice), and KC mice with disruption of Prkd1 (PRKD1KO-KC mice). Subcutaneous xenograft tumors were grown in NSG mice from Panc1 cells; some mice were then given injections of sEVs. Pancreata and lung tissues from mice were analyzed by histology, immunohistochemistry, and/or quantitative polymerase chain reaction; we performed nanoparticle tracking analysis of plasma sEVs. The Prkd1 gene was disrupted in Panc1 cells using CRISPR-Cas9 or knocked down with small hairpin RNAs, or PRKD1 activity was inhibited with the selective inhibitor CRT0066101. Pancreatic cancer cell lines were analyzed by gene-expression microarray, quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. sEVs secreted by Panc1 cell lines were analyzed by flow cytometry, transmission electron microscopy, and mass spectrometry. Levels of PRKD1 were reduced in human pancreatic ductal adenocarcinoma tissues compared with nontumor tissues. PRKD1KO-KC mice developed more pancreatic intraepithelial neoplasia, at a faster rate, than KC mice, and had more lung metastases and significantly shorter average survival time. Serum from PRKD1KO-KC mice had increased levels of sEVs compared with KC mice. Pancreatic cancer cells with loss or inhibition of PRKD1 increased secretion of sEVs; loss of PRKD1 reduced phosphorylation of its substrate, cortactin, resulting in increased F-actin levels at the plasma membrane. sEVs from cells with loss or reduced expression of PRKD1 had altered content, and injection of these sEVs into mice increased metastasis of xenograft tumors to lung, compared with sEVs from pancreatic cells that expressed PRKD1. PRKD1-deficient pancreatic cancer cells showed increased loading of integrin α6β4 into sEVs-a process that required CD82. Human pancreatic ductal adenocarcinoma has reduced levels of PRKD1 compared with nontumor pancreatic tissues. Loss of PRKD1 results in reduced phosphorylation of cortactin in pancreatic cancer cell lines, resulting in increased in F-actin at the plasma membrane and increased release of sEVs, with altered content. These sEVs promote metastasis of xenograft and pancreatic tumors to lung in mice.

Atorvastatin attenuates vascular remodelling in spontaneously hypertensive rats via the protein kinase D/extracellular signal-regulated kinase 5 pathway.

The present study was conducted to determine whether atorvastatin reduces hypertension-induced vascular remodelling and whether its effects involve protein kinase D (PKD) and extracellular signal-regulated kinase 5 (ERK5). We used 16-week-old spontaneously hypertensive rats (SHRs) and age-matched Wistar-Kyoto (WKY) rats. The blood pressure and serum lipid concentration were measured. Changes in the vascular morphology and histology were examined using H&E, Masson' s trichrome, and Sirius Red staining. The media thickness (MT), ratio of MT to lumen diameter (LD) (MT/LD), collagen volume fraction (CVF) and hydroxyproline content were measured to evaluate vascular remodelling. Atorvastatin (50mg/kg/day) was administered for 8weeks. Increased blood pressure and vascular remodelling were more prominent in SHRs than in WKY rats. SHRs also had elevated PKD and ERK5 activation. The systolic blood pressure, MT/LD ratio, and hydroxyproline content were positively correlated with the activation level of PKD and ERK5 in SHRs. Atorvastatin significantly attenuated the activation of PKD and ERK5. Overall, this study demonstrated that atorvastatin could reverse vascular remodelling in SHRs. The PKD/ERK5 signalling pathway might be important for elucidating the beneficial pleiotropic effects of atorvastatin on vascular remodelling.

It Takes Two to Tango: Activation of Protein Kinase D by Dimerization.

The recent discovery and structure determination of a novel ubiquitin-like dimerization domain in protein kinase D (PKD) has significant implications for its activation. PKD is a serine/threonine kinase activated by the lipid second messenger diacylglycerol (DAG). It is an essential and highly conserved protein that is implicated in plasma membrane directed trafficking processes from the trans-Golgi network. However, many open questions surround its mechanism of activation, its localization, and its role in the biogenesis of cargo transport carriers. In reviewing this field, the focus is primarily on the mechanisms that control the activation of PKD at precise locations in the cell. In light of the new structural findings, the understanding of the mechanisms underlying PKD activation is critically evaluated, with particular emphasis on the role of dimerization in PKD autophosphorylation, and the provenance and recognition of the DAG that activates PKD.

Ameliorating effects of Gö6976, a pharmacological agent that inhibits protein kinase D, on collagen-induced arthritis.

Toll-like receptor (TLR) signaling can contribute to the pathogenesis of arthritis. Disruption of TLR signaling at early stages of arthritis might thereby provide an opportunity to halt the disease progression and ameliorate outcomes. We previously found that Gö6976 inhibits TLR-mediated cytokine production in human and mouse macrophages by inhibiting TLR-dependent activation of protein kinase D1 (PKD1), and that PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs. In this study, we investigated whether PKD1 contributes to TLR-mediated proinflammatory responses in human synovial cells, and whether Gö6976 treatment can suppress the development and progression of type II collagen (CII)-induced arthritis (CIA) in mouse. We found that TLR/IL-1R ligands induced activation of PKD1 in human fibroblast-like synoviocytes (HFLS). TLR/IL-1R-induced expression of cytokines/chemokines was substantially inhibited in Gö6976-treated HFLS and PKD1-knockdown HFLS. In addition, serum levels of anti-CII IgG antibodies, and the incidence and severity of arthritis after CII immunization were significantly reduced in mice treated daily with Gö6976. Synergistic effects of T-cell receptor and TLR, as well as TLR alone, on spleen cell proliferation and cytokine production were significantly inhibited in the presence of Gö6976. Our results suggest a possibility that ameliorating effects of Gö6976 on CIA may be due to its ability to inhibit TLR/IL-1R-activated PKD1, which might play an important role in proinflammatory responses in arthritis, and that PKD1 could be a therapeutic target for inflammatory arthritis.

A role for PKD1 in insulin secretion downstream of P2Y1 receptor activation in mouse and human islets.

Along with insulin, β‐cells co‐secrete the neurotransmitter ATP which acts as a positive autocrine signal via P2Y1 receptors to activate phospholipase C and increase the production of diacylglycerol (DAG). However, the downstream signaling that couples P2Y1 activation to insulin secretion remains to be fully elucidated. Since DAG activates protein kinase D1 (PKD1) to potentiate glucose‐stimulated insulin release, we hypothesized that autocrine ATP signaling activates downstream PKD1 to regulate insulin secretion. Indeed, we find that the P2Y1 receptor agonists, MRS2365 and ATP induce, PKD1 phosphorylation at serine 916 in mouse islets. Similarly, direct depolarization of islets by KCl caused PKD1 activation, which is reduced upon P2Y1 antagonism. Potentiation of insulin secretion by P2Y1 activation was lost from PKD1−/− mouse islets, and knockdown of PKD1 reduced the ability of P2Y1 activation to facilitate exocytosis in single mouse β‐cells. Finally, qPCR analysis confirmed PKD1 transcript (PRKD1) expression in human islets, and insulin secretion assays showed that inhibition of either P2Y1 or PKD1 signaling impaired glucose‐stimulated insulin secretion. Human islets showed donor‐to‐donor variation in their responses to both P2Y1 and PKD1 inhibition, however, and we find that the P2Y1‐PKD1 pathway contributes a substantially greater proportion of insulin secretion from islets of overweight and obese donors. Thus, PKD1 promotes increased insulin secretion, likely mediating an autocrine ATP effect via P2Y1 receptor activation which may be more important in islets of donors who are overweight or obese.