Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium.

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

An extensive targeted proteomic analysis of disease-related protein biomarkers in urine from healthy donors.

The analysis of protein biomarkers in urine is expected to lead to advances in a variety of clinical settings. Several characteristics of urine including a low-protein matrix, ease of testing and a demonstrated proteomic stability offer distinct advantages over current widely used biofluids, serum and plasma. Improvements in our understanding of the urine proteome and in methods used in its evaluation will facilitate the clinical development of urinary protein biomarkers. Multiplexed bead-based immunoassays were utilized to evaluate 211 proteins in urines from 103 healthy donors. An additional 25 healthy donors provided serial urine samples over the course of two days in order to assess temporal variation in selected biomarkers. Nearly one-third of the evaluated biomarkers were detected in urine at levels greater than 1 ng/ml, representing a diverse panel of proteins with respect to structure, function and biological role. The presence of several biomarkers in urine was confirmed by western blot. Several methods of data normalization were employed to assess impact on biomarker variability. A complex pattern of correlations with urine creatinine, albumin and beta-2-microglobulin was observed indicating the presence of highly specific mechanisms of renal filtration. Further investigation of the urinary protein biomarkers identified in this preliminary study along with a consideration of the underlying proteomic trends suggested by these findings should lead to an improved capability to identify candidate biomarkers for clinical development.

Common genetic variants in the CLDN2 and PRSS1-PRSS2 loci alter risk for alcohol-related and sporadic pancreatitis.

Pancreatitis is a complex, progressively destructive inflammatory disorder. Alcohol was long thought to be the primary causative agent, but genetic contributions have been of interest since the discovery that rare PRSS1, CFTR and SPINK1 variants were associated with pancreatitis risk. We now report two associations at genome-wide significance identified and replicated at PRSS1-PRSS2 (P < 1 × 10(-12)) and X-linked CLDN2 (P < 1 × 10(-21)) through a two-stage genome-wide study (stage 1: 676 cases and 4,507 controls; stage 2: 910 cases and 4,170 controls). The PRSS1 variant likely affects disease susceptibility by altering expression of the primary trypsinogen gene. The CLDN2 risk allele is associated with atypical localization of claudin-2 in pancreatic acinar cells. The homozygous (or hemizygous in males) CLDN2 genotype confers the greatest risk, and its alleles interact with alcohol consumption to amplify risk. These results could partially explain the high frequency of alcohol-related pancreatitis in men (male hemizygote frequency is 0.26, whereas female homozygote frequency is 0.07).

ADP-ribosylation factor 1 protein regulates trypsinogen activation via organellar trafficking of procathepsin B protein and autophagic maturation in acute pancreatitis.

Several studies have suggested that autophagy might play a deleterious role in acute pancreatitis via intra-acinar activation of digestive enzymes. The prototype for this phenomenon is cathepsin B-mediated trypsin generation. To determine the organellar basis of this process, we investigated the subcellular distribution of the cathepsin B precursor, procathepsin B. We found that procathepsin B is enriched in Golgi-containing microsomes, suggesting a role for the ADP-ribosylation (ARF)-dependent trafficking of cathepsin B. Indeed, caerulein treatment increased processing of procathepsin B, whereas a known ARF inhibitor brefeldin A (BFA) prevented this. Similar treatment did not affect processing of procathepsin L. BFA-mediated ARF1 inhibition resulted in reduced cathepsin B activity and consequently reduced trypsinogen activation. However, formation of light chain 3 (LC3-II) was not affected, suggesting that BFA did not prevent autophagy induction. Instead, sucrose density gradient centrifugation and electron microscopy showed that BFA arrested caerulein-induced autophagosomal maturation. Therefore, ARF1-dependent trafficking of procathepsin B and the maturation of autophagosomes results in cathepsin B-mediated trypsinogen activation induced by caerulein.

Lipotoxicity causes multisystem organ failure and exacerbates acute pancreatitis in obesity.

Obesity increases the risk of adverse outcomes during acute critical illnesses such as burns, severe trauma, and acute pancreatitis. Although individuals with more body fat and higher serum cytokines and lipase are more likely to experience problems, the roles that these characteristics play are not clear. We used severe acute pancreatitis as a representative disease to investigate the effects of obesity on local organ function and systemic processes. In obese humans, we found that an increase in the volume of intrapancreatic adipocytes was associated with more extensive pancreatic necrosis during acute pancreatitis and that acute pancreatitis was associated with multisystem organ failure in obese individuals. In vitro studies of pancreatic acinar cells showed that unsaturated fatty acids were proinflammatory, releasing intracellular calcium, inhibiting mitochondrial complexes I and V, and causing necrosis. Saturated fatty acids had no such effects. Inhibition of lipolysis in obese (ob/ob) mice with induced pancreatitis prevented a rise in serum unsaturated fatty acids and prevented renal injury, lung injury, systemic inflammation, hypocalcemia, reduced pancreatic necrosis, and mortality. Thus, therapeutic approaches that target unsaturated fatty acid-mediated lipotoxicity may reduce adverse outcomes in obese patients with critical illnesses such as severe acute pancreatitis.

Transcriptional regulation of CXC-ELR chemokines KC and MIP-2 in mouse pancreatic acini.

Neutrophils and their chemoattractants, the CXC-ELR chemokines keratinocyte cytokine (KC) and macrophage inflammatory protein-2 (MIP-2), play a critical role in pancreatitis. While acute pancreatitis is initiated in acinar cells, it is unclear if these are a source of CXC-ELR chemokines. KC and MIP-2 have NF-κB, activator protein-1 (AP-1) sites in their promoter regions. However, previous studies have shown increased basal and reduced caerulein-induced AP-1 activation in harvested pancreatic tissue in vitro, which limits interpreting the caerulein-induced response. Moreover, recent studies suggest that NF-κB silencing in acinar cells alone may not be sufficient to reduce inflammation in acute pancreatitis. Thus the aim of this study was to determine whether acinar cells are a source of KC and MIP-2 and to understand their transcriptional regulation. Primary overnight-cultured murine pancreatic acini were used after confirming their ability to replicate physiological and pathological acinar cell responses. Upstream signaling resulting in KC, MIP-2 upregulation was studied along with activation of the transcription factors NF-κB and AP-1. Cultured acini replicated critical responses to physiological and pathological caerulein concentrations. KC and MIP-2 mRNA levels increased in response to supramaximal but not to physiological caerulein doses. This upregulation was calcium and protein kinase C (PKC), but not cAMP, dependent. NF-κB inhibition completely prevented upregulation of KC but not MIP-2. Complete suppression of MIP-2 upregulation required dual inhibition of NF-κB and AP-1. Acinar cells are a likely source of KC and MIP-2 upregulation during pancreatitis. This upregulation is dependent on calcium and PKC. MIP-2 upregulation requires both NF-κB and AP-1 in these cells. Thus dual inhibition of NF-κB and AP-1 may be a more successful strategy to reduce inflammation in pancreatitis than targeting NF-κB alone.

The 19-amino acid insertion in the tumor-associated splice isoform Rac1b confers specific binding to p120 catenin.

The Rac1b splice isoform contains a 19-amino acid insertion not found in Rac1; this insertion leads to decreased GTPase activity and reduced affinity for GDP, resulting in the intracellular predominance of GTP-bound Rac1b. Here, using co-precipitation and proteomic methods, we find that Rac1b does not bind to many common regulators of Rho family GTPases but that it does display enhanced binding to SmgGDS, RACK1, and p120 catenin (p120(ctn)), proteins involved in cell-cell adhesion, motility, and transcriptional regulation. We use molecular modeling and structure analysis approaches to determine that the interaction between Rac1b and p120(ctn) is dependent upon protein regions that are predicted to be unstructured in the absence of molecular complex formation, suggesting that the interaction between these two proteins involves coupled folding and binding. We also find that directed cell movement initiated by Rac1b is dependent upon p120. These results define a distinct binding functionality of Rac1b and provide insight into how the distinct phenotypic program activated by this protein may be implemented through molecular recognition of effectors distinct from those of Rac1.

Caveolae mediate growth factor-induced disassembly of adherens junctions to support tumor cell dissociation.

Remodeling of cell-cell contacts through the internalization of adherens junction proteins is an important event during both normal development and the process of tumor cell metastasis. Here we show that the integrity of tumor cell-cell contacts is disrupted after epidermal growth factor (EGF) stimulation through caveolae-mediated endocytosis of the adherens junction protein E-cadherin. Caveolin-1 and E-cadherin closely associated at cell borders and in internalized structures upon stimulation with EGF. Furthermore, preventing caveolae assembly through reduction of caveolin-1 protein or expression of a caveolin-1 tyrosine phospho-mutant resulted in the accumulation of E-cadherin at cell borders and the formation of tightly adherent cells. Most striking was the fact that exogenous expression of caveolin-1 in tumor cells that contain tight, well-defined, borders resulted in a dramatic dispersal of these cells. Together, these findings provide new insights into how cells might disassemble cell-cell contacts to help mediate the remodeling of adherens junctions, and tumor cell metastasis and invasion.

Matrix metalloproteinases stimulate epithelial-mesenchymal transition during tumor development.

Matrix metalloproteinases (MMPs) are a family of more than 28 enzymes that were initially identified on the basis of their ability to cleave most elements of the extracellular matrix (ECM) but have subsequently been found to be upregulated in nearly every tumor type. As digestion of the ECM is essential for tumor invasion and metastasis, MMPs have been studied for their role in these later stages of tumor development. More recently, exposure to these enzymes has been found to impact cellular signaling pathways that stimulate cell growth at early stages of tumor progression. MMPs have also been found to cleave intracellular targets and so inducing mitotic abnormalities and genomic instability. Emerging evidence indicates that tumor-associated MMPs can also stimulate processes associated with epithelial-mesenchymal transition (EMT), a developmental process that is activated in tumor cells during cell invasion and metastasis. Investigations of potential therapeutic MMP inhibitors aimed at blocking the protumorigenic tissue alterations induced by MMPs have been complicated by the side effects associated with nonspecific inhibition of normal physiological processes; recent investigations have shown how delineation of the extracellular targets and intracellular signaling pathways by which MMP action on cancer cells can induce EMT provides insight into novel therapeutic targets. Here, we provide an overview of recent findings of MMP action in tumors and the mechanisms by which MMPs induce both phenotypic and genotypic alterations that facilitate tumor progression.

Epithelial growth factor-induced phosphorylation of caveolin 1 at tyrosine 14 stimulates caveolae formation in epithelial cells.

Caveolae are flask-shaped endocytic structures composed primarily of caveolin-1 (Cav1) and caveolin-2 (Cav2) proteins. Interestingly, a cytoplasmic accumulation of Cav1 protein does not always result in a large number of assembled caveolae organelles, suggesting a regulatory mechanism that controls caveolae assembly. In this study we report that stimulation of epithelial cells with epithelial growth factor (EGF) results in a profound increase in the number of caveolar structures at the plasma membrane. Human pancreatic tumor cells (PANC-1) and normal rat kidney cells (NRK), as a control, were treated with 30 ng/ml EGF for 0, 5, and 20 min before fixation and viewing by electron microscopy. Cells fixed without EGF treatment exhibited modest numbers of plasma membrane-associated caveolae. Cells treated with EGF for 5 or 20 min showed an 8-10-fold increase in caveolar structures, some forming long, pronounced caveolar "towers" at the cell-cell borders. It is known that Cav1 is Src-phosphorylated on tyrosine 14 in response to EGF treatment, although the significance of this modification is unknown. We postulated that phosphorylation could provide the stimulus for caveolae assembly. To this end, we transfected cells with mutant forms of Cav1 that could not be phosphorylated (Cav1Y14F) and tested if this altered protein reduced the number of EGF-induced caveolae. We observed that EGF-stimulated PANC-1 cells expressing the mutant Cav1Y14F protein exhibited a 90-95% reduction in caveolae number compared with cells expressing wild type Cav1. This study provides novel insights into how cells regulate caveolae formation and implicates EGF-based signaling cascades in the phosphorylation of Cav1 as a stimulus for caveolae assembly.