The genetic risk factor CEL-HYB1 causes proteotoxicity and chronic pancreatitis in mice.

BACKGROUND & AIMS: The CEL gene encodes the digestive enzyme carboxyl ester lipase. CEL-HYB1, a hybrid allele of CEL and its adjacent pseudogene CELP, is a genetic variant suggested to increase the risk of chronic pancreatitis (CP). Our aim was to develop a mouse model for CEL-HYB1 that enables studies of pancreatic disease mechanisms. METHODS: We established a knock-in mouse strain where the variable number of tandem repeat (VNTR) region of the endogenous mouse Cel gene was substituted with the mutated VNTR of the human CEL-HYB1 allele. Heterozygous and homozygous Cel-HYB1 mice and littermate wildtype controls were characterized with respect to pancreatic pathology and function. RESULTS: We successfully constructed a mouse model with pancreatic expression of a humanized CEL-HYB1 protein. The Cel-HYB1 mice spontaneously developed features of CP including inflammation, acinar atrophy and fatty replacement, and the phenotype became more pronounced as the animals aged. Moreover, Cel-HYB1 mice were normoglycemic at age 6 months, whereas at 12 months they exhibited impaired glucose tolerance. Immunostaining of pancreatic tissue indicated the formation of CEL protein aggregates, and electron microscopy showed dilated endoplasmic reticulum. Upregulation of the stress marker BiP/GRP78 was seen in pancreatic parenchyma obtained both from Cel-HYB1 animals and from a human CEL-HYB1 carrier. CONCLUSIONS: We have developed a new mouse model for CP that confirms the pathogenicity of the human CEL-HYB1 variant. Our findings place CEL-HYB1 in the group of genes that increase CP risk through protein misfolding-dependent pathways.

Integrin α11ß1 and syndecan-4 dual receptor ablation attenuate cardiac hypertrophy in the pressure overloaded heart.

Pathological myocardial hypertrophy in response to an increase in left ventricular (LV) afterload may ultimately lead to heart failure. Cell surface receptors bridge the interface between the cell and the extracellular matrix (ECM) in cardiac myocytes and cardiac fibroblasts and have been suggested to be important mediators of pathological myocardial hypertrophy. We identify for the first time that integrin α11 (α11) is preferentially upregulated among integrin ß1 heterodimer-forming α-subunits in response to increased afterload induced by aortic banding (AB) in wild-type (WT) mice. Mice were anesthetized in a chamber with 4% isoflurane and 95% oxygen before being intubated and ventilated with 2.5% isoflurane and 97% oxygen. For pre- and postoperative analgesia, animals were administered 0.02-mL buprenorphine (0.3 mg/mL) subcutaneously. Surprisingly, mice lacking α11 develop myocardial hypertrophy following AB comparable to WT. In the mice lacking α11, we further show a compensatory increase in the expression of another mechanoreceptor, syndecan-4, following AB compared with WT AB mice, indicating that syndecan-4 compensated for lack of α11. Intriguingly, mice lacking mechanoreceptors α11 and syndecan-4 show ablated myocardial hypertrophy following AB compared with WT mice. Expression of the main cardiac collagen isoforms col1a2 and col3a1 was significantly reduced in AB mice lacking mechanoreceptors α11 and syndecan-4 compared with WT AB.NEW & NOTEWORTHY Despite their putative importance in stress sensing, the specific integrin α-subunit(s) involved in cardiac hypertrophy has not been identified. Here, we show that α11 and syndecan-4 are critical and interdependent mediators of the hypertrophic response to increased LV afterload. We demonstrate in cells lacking both receptors an interdependent reduction in cell attachment to the major cardiac extracellular matrix components, suggesting that their interplay represents an important mechanism for stress sensing in cardiac cells.

Abnormal exocrine-endocrine cell cross-talk promotes ß-cell dysfunction and loss in MODY8.

MODY8 (maturity-onset diabetes of the young, type 8) is a dominantly inherited monogenic form of diabetes associated with mutations in the carboxyl ester lipase (CEL) gene expressed by pancreatic acinar cells. MODY8 patients develop childhood-onset exocrine pancreas dysfunction followed by diabetes during adulthood. However, it is unclear how CEL mutations cause diabetes. In the present study, we report the transfer of CEL proteins from acinar cells to ß-cells as a form of cross-talk between exocrine and endocrine cells. Human ß-cells show a relatively higher propensity for internalizing the mutant versus the wild-type CEL protein. After internalization, the mutant protein forms stable intracellular aggregates leading to ß-cell secretory dysfunction. Analysis of pancreas sections from a MODY8 patient reveals the presence of CEL protein in the few extant ß-cells. The present study provides compelling evidence for the mechanism by which a mutant gene expressed specifically in acinar cells promotes dysfunction and loss of ß-cells to cause diabetes.

Generation of a novel mouse strain with fibroblast-specific expression of Cre recombinase.

Cell-specific expression of genes offers the possibility to use their promoters to drive expression of Cre-recombinase, thereby allowing for detailed expression analysis using reporter gene systems, cell lineage tracing, conditional gene deletion, and cell ablation. In this context, current data suggest that the integrin α11 subunit has the potential to serve as a fibroblast biomarker in tissue regeneration and pathology, in particular in wound healing and in tissue- and tumor fibrosis. The mesenchyme-restricted expression pattern of integrin α11 thus prompted us to generate a novel ITGA11-driver Cre mouse strain using a ϕC31 integrase-mediated knock-in approach. In this transgenic mouse, the Cre recombinase is driven by regulatory promoter elements within the 3 kb segment of the human ITGA11 gene. ß-Galactosidase staining of embryonic tissues obtained from a transgenic ITGA11-Cre mouse line crossed with Rosa 26R reporter mice (ITGA11-Cre;R26R) revealed ITGA11-driven Cre expression and activity in mesenchymal cells in a variety of mesenchymal tissues in a pattern reminiscent of endogenous α11 protein expression in mouse embryos. Interestingly, X-gal staining of mouse embryonic fibroblasts (MEFs) isolated from the ITGA11-Cre;R26R mice indicated heterogeneity in the MEF population. ITGA11-driven Cre activity was shown in approximately 60% of the MEFs, suggesting that the expression of integrin α11 could be exploited for isolation of different fibroblast populations. ITGA11-driven Cre expression was found to be low in adult mouse tissues but was induced in granulation tissue of excisional wounds and in fibrotic hearts following aortic banding. We predict that the ITGA11-Cre transgenic mouse strain described in this report will be a useful tool in matrix research for the deletion of genes in subsets of fibroblasts in the developing mouse and for determining the function of subsets of pro-fibrotic fibroblasts in tissue fibrosis and in different subsets of cancer-associated fibroblasts in the tumor microenvironment.

Cancer-associated fibroblasts in desmoplastic tumors: emerging role of integrins.

The tumor microenvironment (TME) is a complex meshwork of extracellular matrix (ECM) macromolecules filled with a collection of cells including cancer-associated fibroblasts (CAFs), blood vessel associated smooth muscle cells, pericytes, endothelial cells, mesenchymal stem cells and a variety of immune cells. In tumors the homeostasis governing ECM synthesis and turnover is disturbed resulting in abnormal blood vessel formation and excessive fibrillar collagen accumulations of varying stiffness and organization. The disturbed ECM homeostasis opens up for new types of paracrine, cell-cell and cell-ECM interactions with large consequences for tumor growth, angiogenesis, metastasis, immune suppression and resistance to treatments. As a main producer of ECM and paracrine signals the CAF is a central cell type in these events. Whereas the paracrine signaling has been extensively studied in the context of tumor-stroma interactions, the nature of the numerous integrin-mediated cell-ECM interactions occurring in the TME remains understudied. In this review we will discuss and dissect the role of known and potential CAF interactions in the TME, during both tumorigenesis and chemoresistance-induced events, with a special focus on the "interaction landscape" in desmoplastic breast, lung and pancreatic cancers. As an example of the multifaceted mode of action of the stromal collagen receptor integrin α11ß1, we will summarize our current understanding on the role of this CAF-expressed integrin in these three tumor types.

Integrin α11 cytoplasmic tail is required for FAK activation to initiate 3D cell invasion and ERK-mediated cell proliferation.

Integrin α11ß1 is a collagen-binding integrin, which is receiving increasing attention in the context of wound healing and fibrosis. Although α11ß1 integrin displays similar collagen specificity to α2ß1 integrin, both integrins have distinct in vivo functions. In this context, the contribution of α11 subunit cytoplasmic tail interactions to diverse molecular signals and biological functions is largely unknown. In the current study, we have deleted the α11 cytoplasmic tail and studied the effect of this deletion on α11 integrin function. Compared to wild-type cells, C2C12 cells expressing tail-less α11 attached normally to collagen I, but formed fewer focal contacts. α11-tail-less cells furthermore displayed a reduced capacity to invade and reorganize a 3D collagen matrix and to proliferate. Analysis of cell signaling showed that FAK and ERK phosphorylation was reduced in cells expressing tail-less α11. Inhibition of ERK and FAK activation decreased α11-mediated cell proliferation, whereas α11-mediated cell invasion was FAK-dependent and occurred independently of ERK signaling. In summary, our data demonstrate that the integrin α11 cytoplasmic tail plays a central role in α11 integrin-specific functions, including FAK-dependent ERK activation to promote cell proliferation.

α11ß1 Integrin is Induced in a Subset of Cancer-Associated Fibroblasts in Desmoplastic Tumor Stroma and Mediates In Vitro Cell Migration.

Integrin α11ß1 is a collagen receptor that has been reported to be overexpressed in the stroma of non-small cell lung cancer (NSCLC) and of head and neck squamous cell carcinoma (HNSCC). In the current study, we further analyzed integrin α11 expression in 14 tumor types by screening a tumor tissue array while using mAb 203E3, a newly developed monoclonal antibody to human α11. Different degrees of expression of integrin α11 were observed in the stroma of breast, ovary, skin, lung, uterus, stomach, and pancreatic ductal adenocarcinoma (PDAC) tumors. Co-expression queries with the myofibroblastic cancer-associated fibroblast (myCAF) marker, alpha smooth muscle actin (αSMA), demonstrated a moderate level of α11+ in myCAFs associated with PDAC and HNSCC tumors, and a lack of α11 expression in additional stromal cells (i.e., cells positive for fibroblast-specific protein 1 (FSP1) and NG2). The new function-blocking α11 antibody, mAb 203E1, inhibited cell adhesion to collagen I, partially hindered fibroblast-mediated collagen remodeling and obstructed the three-dimensional (3D) migration rates of PDAC myCAFs. Our data demonstrate that integrin α11 is expressed in a subset of non-pericyte-derived CAFs in a range of cancers and suggest that α11ß1 constitutes an important receptor for collagen remodeling and CAF migration in the tumor microenvironment (TME).

The long non-coding RNA lnc-ZNF180-2 is a prognostic biomarker in patients with clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is among the most common human malignancies. Long non-coding RNAs (lncRNA) regulate various cellular functions and have been implicated in ccRCC pathogenesis. In order to decipher the molecular biology of this tumor and to identify potential prognostic biomarkers and therapeutic targets, we re-evaluated published lncRNA expression profiling data. An expression profile of 49 lncRNAs allowed discrimination of localized and advanced ccRCC. The expression profile of six lncRNAs transcripts (lnc-ACO1625, lnc-CYP4A22-2/3, lnc-PEAK1.1-1, lnc-PCYOX1L, lnc-VCAN-1, lnc-ZNF180-2) with potential prognostic interest were validated in a cohort of 50 normal renal, 57 localized ccRCC and 45 advanced ccRCC tissues. lnc-ZNF180-2 levels were similar in localized ccRCC and normal renal tissue, but we observed a significant increase of lnc-ZNF180-2 expression in advanced ccRCC tissue. Furthermore, lnc-ZNF180-2 expression levels were an independent predictor of progression-free survival, cancer-specific survival and overall survival in ccRCC patients. We also observed that lnc-CYP4A22-2/3 expression levels allowed discrimination of ccRCC and normal renal tissue. In conclusion, lncRNAs are involved in renal carcinogenesis, and quantification of lnc-ZNF180-2 may be useful for the prediction of ccRCC patients outcome following nephrectomy.