PKD1 inhibits cancer cells migration and invasion via Wnt signaling pathway in vitro

The approximately 14 kb mRNA of the polycystic kidney disease gene PKD1 encodes a large ( approximately 460 kDa) protein, termed polycystin-1 (PC-1), that is responsible for autosomal dominant polycystic kidney disease (ADPKD). The unique organization of its multiple adhesive domains (16 Ig-like domains/PKD domains) suggests that it may play an important role in cell-cell/cell-matrix interactions. Here we demonstrated that PKD1 promoted cell-cell and cell-matrix interactions in cancer cells, indicating that PC-1 is involved in the cell adhesion process. Furthermore in this study, we showed that PKD1 inhibited cancer cells migration and invasion. And we also showed that PC-1 regulated these processes in a process that may be at least partially through the Wnt pathway. Collectively, our data suggest that PKD1 may act as a novel member of the tumor suppressor family of genes.     

Off-response property of an acid-activated cation channel complex PKD1L3-PKD2L1

Ligand-gated ion channels are important in sensory and synaptic transduction. The PKD1L3-PKD2L1 channel complex is a sour taste receptor candidate that is activated by acids. Here, we report that the proton-activated PKD1L3-PKD2L1 ion channels have the unique ability to be activated after the removal of an acid stimulus. We refer to this property as the off-response (previously described as a delayed response). Electrophysiological analyses show that acid-induced responses are observed only after the removal of an acid solution at less than pH 3.0. A small increase in pH is sufficient for PKD1L3-PKD2L1 channel activation, after exposure to an acid at pH 2.5. These results indicate that this channel is a new type of ion channel-designated as an 'off-channel'-which is activated during stimulus application but not gated open until the removal of the stimulus. The off-response property of PKD1L3-PKD2L1 channels might explain the physiological phenomena occurring during sour taste sensation.     

Downregulation of PKD1 by shRNA results in defective osteogenic differentiation via cAMP/PKA pathway in human MG-63 cells

Mutations and/or deletions of Pkd1 in mouse models resulted in attenuation of osteoblast function and defective bone formation; however, the function of PKD1 in human osteoblast and bone remains uncertain. In the current study, we used lentivirus-mediated shRNA technology to stably knock down PKD1 in the human osteoblastic MG-63 cell line and to investigate the role of PKD1 on human osteoblast function and molecular mechanisms. We found that a 53% reduction of PKD1 by PKD1 shRNA in stable, transfected MG-63 cells resulted in increased cell proliferation and impaired osteoblastic differentiation as reflected by increased BrdU incorporation, decreased alkaline phosphatase activity, and calcium deposition and by decreased expression of RUNX2 and OSTERIX compared to control shRNA MG-63 cells. In addition, knockdown of PKD1 mRNA caused enhanced adipogenesis in stable PKD1 shRNA MG-63 cells as evidenced by elevated lipid accumulation and increased expression of adipocyte-related markers such as PPARγ and aP2. The stable PKD1 shRNA MG-63 cells exhibited lower basal intracellular calcium, which led to attenuated cytosolic calcium signaling in response to fluid flow shear stress, as well as increased intracellular cAMP messages in response to forskolin (10 μM) stimulation. Moreover, increased cell proliferation, inhibited osteoblastic differentiation, and osteogenic and adipogenic gene markers were significantly reversed in stable PKD1 shRNA MG-63 cells when treated with H89 (1 μM), an inhibitor of PKA. These findings suggest that downregulation of PKD1 in human MG-63 cells resulted in defective osteoblast function via intracellular calcium-cAMP/PKA signaling pathway.     

Acetic acid activates PKD1L3-PKD2L1 channel—A candidate sour taste receptor

The polycystic kidney disease (PKD) 1L3-PKD2L1 channel is a candidate sour taste receptor expressed in mammalian taste receptor cells. Various acids are reported to activate PKD channels after the removal of the acid stimuli, but little information is available on the activation of these channels by acetic acid. It was difficult to analyze the PKD channel activation by acetic acid using Ca²⁺ imaging experiments because this acid induces a transient and nonspecific response in cultured cells. Here, we developed a novel method to evaluate PKD channel activation by acetic acid. Nonspecific responses were observed only over a short period after the application of acetic acid. In contrast, PKD channel activation evoked by acetic acid as well as citric acid was detected even at a later time point. This method revealed that PKD1L3-PKD2L1 channel activation by acetic acid was pH-dependent and occurred when the ambient pH was <3.1.     

RBM10 inhibits cell proliferation of lung adenocarcinoma via RAP1/AKT/CREB signalling pathway

Initial functional studies have demonstrated that RNA‐binding motif protein 10 (RBM10) can promote apoptosis and suppress cell proliferation; however, the results of several studies suggest a tumour‐promoting role for RBM10. Herein, we assessed the involvement of RBM10 in lung adenocarcinoma cell proliferation and explored the potential molecular mechanism. We found that, both in vitro and in vivo, RBM10 overexpression suppresses lung adenocarcinoma cell proliferation, while its knockdown enhances cell proliferation. Using complementary DNA microarray analysis, we previously found that RBM10 overexpression induces significant down‐regulation of RAP1A expression. In this study, we have confirmed that RBM10 decreases the activation of RAP1 and found that EPAC stimulation and inhibition can abolish the effects of RBM10 knockdown and overexpression, respectively, and regulate cell growth. This effect of RBM10 on proliferation was independent of the MAPK/ERK and P38/MAPK signalling pathways. We found that RBM10 reduces the phosphorylation of CREB via the AKT signalling pathway, suggesting that RBM10 exhibits its effect on lung adenocarcinoma cell proliferation via the RAP1/AKT/CREB signalling pathway.     

miR-483 inhibits bovine myoblast cell proliferation and differentiation via IGF1/PI3K/AKT signal pathway

MicroRNAs (miRNAs) have been established to regulate skeletal muscle development in mammals. However, few studies have been conducted on the regulation of proliferation and differentiation of bovine myoblast cells by miRNAs. The aim of our study was to explore the function of miR-483 in cell proliferation and differentiation of bovine myoblast. Here, we found that miR-483 declined in both proliferation and differentiation stages of bovine myoblast cells. During the proliferation phase, the overexpression of miR-483 downregulated the cell cycle–associated genes cyclin-dependent kinase 2 (CDK2), proliferating cell nuclear antigen (PCNA) messenger RNA (mRNA), and the protein levels. At the cellular level, cell cycle, cell counting kit-8, and 5-ethynyl-2´-deoxyuridine results indicated that the overexpression of miR-483 block cell proliferation. During differentiation, the overexpression of miR-483 led to a decrease in the levels of the myogenic marker genes MyoD1 and MyoG mRNA and protein. Furthermore, the immunofluorescence analysis results showed that the number of MyHC-positive myotubes was reduced. In contrast, the opposite experimental results were obtained concerning both proliferation and differentiation after the inhibition of miR-483. Mechanistically, we demonstrated that miR-483 target insulin-like growth factor 1 (IGF1) and downregulated the expression of key proteins in the PI3K/AKT signaling pathway. Altogether, our findings indicate that miR-483 acts as a negative regulator of bovine myoblast cell proliferation and differentiation.     

Retracted: Upregulated microRNA-31 inhibits oxidative stress-induced neuronal injury through the JAK/STAT3 pathway by binding to PKD1 in mice with ischemic stroke

Ischemic stroke (IS), which is characterized by high morbidity, disability, and mortality, is recognized as a major cerebrovascular disease. MicroRNA-31 (miR-31) was reported to participate in the progression of brain disease. The present study was conducted in order to investigate the effect of miR-31 on oxidative stress-induced neuronal injury in IS mice with the involvement of protein kinase D1 (PKD1) and the JAK/STAT3 pathway. C57BL/6J mice were used to establish the middle cerebral artery occlusion (MCAO) model. Astrocytes were transfected with miR-31 mimic, miR-31 inhibitor, si-PKD1, or JAK-STAT3 pathway inhibitor. Following the establishment of an oxygen–glucose deprivation (OGD) model, the astrocytes were cocultured with neuronal OGD. Lower miR-31, higher PKD1 expressions, and activated JAK/STAT3 pathway were found in both the MCAO and OGD models. miR-31 could negatively target PKD1. In an MCAO model, overexpressing miR-31 and silencing PKD1 reduced neuronal injury, cerebral infarct volume, neuron loss, and oxidative stress injury, inhibited the activation of JAK/STAT3 pathway and the expressions of PKD1, interleukin (IL)-1β, IL-6, tumor necrosis factor-α, malondialdehyde, 4-HNE, 8-HOdG, caspase-3, and Bax, but increased the superoxide dismutase content. In the OGD model, overexpression of miR-31 and silencing of PKD1 attenuated oxidative stress-induced neuronal injury, and diminished the lactate dehydrogenase leakage and reactive oxygen species level, accompanied by elevated neuronal viability. These results indicate that miR-31 alleviates inflammatory response as well as an oxidative stress-induced neuronal injury in IS mice by downregulating PKD1 and JAK/STAT3 pathway.     

HNF1B inhibits cell proliferation via repression of SMAD6 expression in prostate cancer

Prostate cancer is the most common malignancy in men in developed countries. In previous study, we identified HNF1B (Hepatocyte Nuclear Factor 1β) as a downstream effector of Enhancer of zeste homolog 2 (EZH2). HNF1B suppresses EZH2‐mediated migration of two prostate cancer cell lines via represses the EMT process by inhibiting SLUG expression. Besides, HNF1B expression inhibits cell proliferation through unknown mechanisms. Here, we demonstrated that HNF1B inhibited the proliferation rate of prostate cancer cells. Overexpression of HNF1B in prostate cancer cells led to the arrest of G1 cell cycle and decreased Cyclin D1 expression. In addition, we re‐explored data from ChIP‐sequencing (ChIP‐seq) and RNA‐sequencing (RNA‐seq), and demonstrated that HNF1B repressed Cyclin D1 via direct suppression of SMAD6 expression. We also identified CDKN2A as a HNF1B‐interacting protein that would contribute to HNF1B‐mediated repression of SMAD6 expression. In summary, we provide the novel mechanisms and evidence in support HNF1B as a tumour suppressor gene for prostate cancer.     

LncRNA COL1A2-AS1 inhibits the scar fibroblasts proliferation via regulating miR-21/Smad7 pathway

lncRNA COL1A2-AS1 (COL1A2 antisense RNA 1), a lncRNA overexpressed in hypertrophic scar, has been demonstrated to be involved in the hypertrophic scar formation. However, the mechanisms of lncRNA COL1A2-AS1 inhibiting the scar fibroblasts proliferation remains not well understood. In this study, we demonstrated that lncRNA COL1A2-AS1 was upregulated in hypertrophic scar tissue and fibroblasts, and suppressed fibroblasts proliferation by promoting Smad7 expression. Furthermore, we found that miR-21 was involved in lncRNA COL1A2-AS1-induced expression of Smad7, by which COL1A2-AS1 acted as endogenous sponge to adsorb miR-21 and in turn regulated Smad7 and a cascade of molecular to play a protective role in hypertrophic scar. In addition, overexpression of miR-21 attenuated COL1A2-AS1-mediated proliferation suppression of hypertrophic scar fibroblasts. In conclusion, our study demonstrated that COL1A2-AS1/miR-21/Smad pathway plays an important role in inhibiting hypertrophic scar formation, and suggested this novel pathway may be a new target for hypertrophic scar treatment.     

Phosphorylation at Ser244 by CK1 determines nuclear localization and substrate targeting of PKD2

Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells.     

Carbon monoxide inhibits T lymphocyte proliferation via caspase-dependent pathway

T lymphocyte activation and proliferation is involved in many pathological processes. We have recently shown that carbon monoxide (CO), an enzymatic product of heme oxygenase-1 (HO-1), confers potent antiproliferative effects in airway and vascular smooth muscle cells. The purpose of this study was to determine whether CO can inhibit T lymphocyte proliferation and then to determine the mechanism by which CO can modulate T lymphocyte proliferation. In the presence of 250 parts per million CO, CD3-activated T lymphocyte proliferation was, remarkably, inhibited by 80% when compared with controls. We observed that the antiproliferative effect of CO in T lymphocytes was independent of the mitogen-activated protein kinase or cGMP signaling pathways, unlike what we demonstrated previously in smooth muscle cells. We demonstrate that CO inhibited caspase-3 and caspase-8 expression and activity, and caspase inhibition with benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK pan-caspase inhibitor) blocked T lymphocyte proliferation. Furthermore, in caspase-8-deficient lymphocytes, the antiproliferative effect of CO was markedly attenuated, further supporting the involvement of caspase-8 in the antiproliferative effects of CO. CO also increased the protein level of p21(Cip1), and CO-mediated inhibition of caspase activity is partially regulated by p21(Cip1). Taken together, these data suggest that CO confers potent antiproliferative effects in CD3-activated T lymphocytes and that these antiproliferative effects in T lymphocytes are mediated by p21(Cip1)-dependent caspase activity, in particular caspase-8, independent of cGMP and mitogen-activated protein kinase signaling pathways.     

Heat shock protein 20 (HSP20) is a novel substrate for protein kinase D1 (PKD1)

Heat shock protein 20 (HSP20) has cardioprotective qualities, which are triggered by PKA phosphorylation. PKD1 is also a binding partner for HSP20, and this prompted us to investigate whether the chaperone was a substrate for PKD1. We delineate the PKD1 binding sites on HSP20 and show for the first time HSP20 is a substrate for PKD1. Phosphorylation of HSP20 by PKD1 is diminished by pharmacological or siRNA reduction of PKD1 activity and is enhanced following PKD1 activation. Our results suggest that both PKA and PKD1 can both phosphorylate HSP20 on serine 16 but that PKA is the most dominant. © 2016 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.     

Regulatory domain determinants that control PKD1 activity

The canonical pathway for protein kinase D1 (PKD1) activation by growth factor receptors involves diacylglycerol binding to the C1 domain and protein kinase C-dependent phosphorylation at the activation loop. PKD1 then autophosphorylates at Ser(916), a modification frequently used as a surrogate marker of PKD1 activity. PKD1 also is cleaved by caspase-3 at a site in the C1-PH interdomain during apoptosis; the functional consequences of this cleavage event remain uncertain. This study shows that PKD1-Δ1-321 (an N-terminal deletion mutant lacking the C1 domain and flanking sequence that models the catalytic fragment that accumulates during apoptosis) and PKD1-CD (the isolated catalytic domain) display high basal Ser(916) autocatalytic activity and robust activity toward CREBtide (a peptide substrate) but little to no activation loop autophosphorylation and no associated activity toward protein substrates, such as cAMP-response element binding protein and cardiac troponin I. In contrast, PKD1-ΔPH (a PH domain deletion mutant) is recovered as a constitutively active enzyme, with high basal autocatalytic activity and high basal activity toward peptide and protein substrates. These results indicate that individual regions in the regulatory domain act in a distinct manner to control PKD1 activity. Finally, cell-based studies show that PKD1-Δ1-321 does not substitute for WT-PKD1 as an in vivo activator of cAMP-response element binding protein and ERK phosphorylation. Proteolytic events that remove the C1 domain (but not the autoinhibitory PH domain) limit maximal PKD1 activity toward physiologically relevant protein substrates and lead to a defect in PKD1-dependent cellular responses.     

hCLP46 regulates U937 cell proliferation via Notch signaling pathway

Human CAP10-like protein 46 kDa (hCLP46) is the homolog of Rumi, which is the first identified protein O-glucosyltransferase that modifies Notch receptor in Drosophila. Dysregulation of hCLP46 occurs in many hematologic diseases, but the role of hCLP46 remains unclear. Knockdown of hCLP46 by RNA interference resulted in decreased protein levels of endogenous Notch1, Notch intracellular domain (NICD) and Notch target gene Hes-1, suggesting the impairment of the Notch signaling. However, neither cell surface Notch expression nor ligand binding activities were affected. In addition, down-regulated expression of hCLP46 inhibited the proliferation of U937 cells, which was correlated with increased cyclin-dependent kinase inhibitor (CDKI) CDKN1B (p27) and decreased phosphorylation of retinoblastoma (RB) protein. We showed that lack of hCLP46 results in impaired ligand induced Notch activation in mammalian cell, and hCLP46 regulates the proliferation of U937 cell through CDKI-RB signaling pathway, which may be important for the pathogenesis of leukemia.