Novel Small Molecule Inhibitors of Protein Kinase D Suppress NF-kappaB Activation and Attenuate the Severity of Rat Cerulein Pancreatitis

Abstract

Nuclear factor-kappa B (NF-κB) activation is a key early signal regulating inflammatory and cell death responses in acute pancreatitis. Our previous in vitro studies with molecular approaches on AR42J cell showed that protein kinase D (PKD/PKD1) activation was required in NF-κB activation induced by cholecystokinin 8 (CCK) or carbachol (CCh) in pancreatic acinar cells. Recently developed small molecule PKD inhibitors, CID755673 and CRT0066101, provide potentially important pharmacological approaches to further investigate the effect of PKD in pancreatitis therapy. The aim of this study was to explore whether CID755673 and CRT0066101 block NF-κB activation with in vitro and in vivo models of experimental pancreatitis and whether the small molecule PKD inhibitors have therapeutic effects when given before or after the initiation of experimental pancreatitis. Freshly prepared pancreatic acini were incubated with CID755673 or CRT006101, followed by hyperstimulation with CCK or CCh. For in vivo experimental pancreatitis, rats were treated with intraperitoneal injection of CID755673 or CRT0066101 prior to or after administering cerulein or saline. PKD activation and NF-κB-DNA binding activity in nuclear extracts from pancreatic acini and tissue were measured. The effects of PKD inhibitors on pancreatitis responses were evaluated. Our results showed that both CID755673 or CRT0066101 selectively and specifically inhibited PKD without effects on related protein kinase Cs. Inhibition of PKD resulted in significantly attenuation of NF-κB activation in both in vitro and in vivo models of experimental pancreatitis. NF-κB inhibition by CID755673 was associated with decreased inflammatory responses and attenuated severity of the disease, which were indicated by less inflammatory cell infiltration, reduced pancreatic interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1), decreased intrapancreatic trypsin activation, and alleviation in pancreatic necrosis, edema and vacuolization. Furthermore, PKD inhibitor CID755673, given after the initiation of pancreatitis in experimental rat model, significantly attenuated the severity of acute pancreatitis. Therapies for acute pancreatitis are limited. Our results indicate that small chemical PKD inhibitors have significant potential as therapeutic interventions by suppressing NF-κB activation.

Protein kinase D mediates inflammatory responses of human placental macrophages to Group B Streptococcus

“…Considering PKD's role in inflammatory activation, it is no surprise that PKD activation has been linked to NFκB signaling. Inhibiting PKD with pharmacological inhibitors resulted in an inhibition of NFκB signaling; consistent with this, the PKD gene (PRKD1) promoter contains binding sites for NFκB [44][45][46]70 . Moreover, GBS infection was shown to downregulate the expression of IκBα in THP-1 macrophages and mouse spleen; IκBα downregulation was reversed using the potent PKC/PKD1 inhibitor Gӧ6976 17 .…”

“…Under oxidative stress and cholinergic signaling, PKD is able to mediate NFκB activation 44,45 . Inhibitors of PKD such as CRT were also shown to attenuate NFκB activation and inflammatory responses in pancreatitis rat models 46 . Placental macrophages were infected with GBS in the presence or absence of CRT to evaluate modulation of NFκB activation, as estimated by the ratio of phosphorylated NFκB p65 to IKBα, since the former will increase as NFκB is activated and the latter will be degraded 47 .…”

Regulation of Osteoclast Differentiation at Multiple Stages by Protein Kinase D Family Kinases

“…Consistent with the hypothesis that CRT0066101's effects on motility are mediated through PKD kinases, we found that 30 µM CID755673 also reduced the number of migrated cells by 55% in the transwell assay compared to the control group, while 10 µM gave a smaller reduction in motility of 31% ( Figure 5C). CID755673 was used at 10 and 30 µM concentrations for this experiment based on our previous studies and consistent with the effective dose against PKD activity in other published cell culture studies [24,[33][34][35]. Taken together, these data suggest that PKD promotes preosteoclast cellular motility.…”

Deciphering the Role of Protein Kinase D1 (PKD1) in Cellular Proliferation

“…Pharmacologic inhibitors of PKD1 attenuated early pancreatitis events (44), but also significantly attenuated pancreatic injury when used as posttreatment (45). These results are of crucial importance because, by promoting pancreatitis-associated necrosis (46), PKD1 would promote a proinflammatory state, especially characterized by the secretion of IL6 and MCP-1 proteins (45), which could give rise to pancreatic lesions, known as risk factors for cancer development. This is consistent with data showing that PKD1 is upregulated in mice pancreatic acinar cells that undergo acinar-to-ductal metaplasia (ADM; ref.…”