Kevin C. Ray

A CK19CreERT Knockin Mouse Line Allows for Conditional DNA Recombination in Epithelial Cells in Multiple Endodermal Organs

Cre/LoxP‐mediated DNA recombination allows for gene function and cell lineage analyses during embryonic development and tissue regeneration. Here, we describe the derivation of a K19CreERT mouse line in which the tamoxifen‐activable CreERT was knocked into the endogenous cytokeratin 19 locus. In the absence of tamoxifen, leaky Cre activity could be detected only in less than 1% of stomach and intestinal epithelial cells, but not in pancreatic or hepatic epithelial tissues. Tamoxifen administration in postnatal animals induced widespread DNA recombination in epithelial cells of pancreatic ducts, hepatic ducts, stomach, and intestine in a dose‐dependent manner. Significantly, we found that Cre activity could be induced in the putative gut stem/progenitor cells that sustained long‐term gut epithelial expression of a Cre reporter. This mouse line should therefore provide a valuable reagent for manipulating gene activity and for cell lineage marking in multiorgans during normal tissue homeostasis and regeneration. genesis 46:318–323, 2008.

Epithelial Tissues Have Varying Degrees of Susceptibility to KrasG12D-Initiated Tumorigenesis in a Mouse Model

Activating mutations in the Kras gene are commonly found in some but not all epithelial cancers. In order to understand the susceptibility of different epithelial tissues to Kras-induced tumorigenesis, we introduced one of the most common Kras mutations, KrasG12D, broadly in epithelial tissues. We used a mouse model in which the G12D mutation is placed in the endogenous Kras locus controlled by inducible, Cre-mediated recombination in tissues expressing cytokeratin 19 including the oral cavity, GI tract, lungs, and ducts of the liver, kidney, and the pancreas. Introduction of the KrasG12D mutation in adult mouse tissues led to neoplastic changes in some but not all of these tissues. Notably, many hyperplasias, metaplasias and adenomas were observed in the oral cavity, stomach, colon and lungs, suggesting that exposure to products of the outside environment promotes KrasG12D-initiated tumorigenesis. However, environmental exposure did not consistently correlate with tumor formation, such as in the small intestine, suggesting that there are also intrinsic differences in susceptibility to Kras activation. The pancreas developed small numbers of mucinous metaplasias with characteristics of early stage pancreatic intraepithelial neoplasms (PanINs), supporting the hypothesis that pancreatic ducts have the potential to give rise pancreatic cancer.

Adult pancreatic acinar cells give rise to ducts but not endocrine cells in response to growth factor signaling

Studies in both humans and rodents have found that insulin+ cells appear within or near ducts of the adult pancreas, particularly following damage or disease, suggesting that these insulin+ cells arise de novo from ductal epithelium. We have found that insulin+ cells are continuous with duct cells in the epithelium that makes up the hyperplastic ducts of both chronic pancreatitis and pancreatic cancer in humans. Therefore, we tested the hypothesis that both hyperplastic ductal cells and their associated insulin+ cells arise from the same cell of origin. Using a mouse model that develops insulin+ cell-containing hyperplastic ducts in response to the growth factor TGFα, we performed genetic lineage tracing experiments to determine which cells gave rise to both hyperplastic ductal cells and duct-associated insulin+ cells. We found that hyperplastic ductal cells arose largely from acinar cells that changed their cell fate, or transdifferentiated, into ductal cells. However, insulin+ cells adjacent to acinar-derived ductal cells arose from pre-existing insulin+ cells, suggesting that islet endocrine cells can intercalate into hyperplastic ducts as they develop. We conclude that apparent pancreatic plasticity can result both from the ability of acinar cells to change fate and of endocrine cells to reorganize in association with duct structures.

Epidermal growth factor receptor regulates pancreatic fibrosis

The development of pancreatic fibrosis has been shown to be a major component in several diseases of the pancreas including pancreatic cancer, chronic pancreatitis, and type 2 diabetes mellitus, but its actual role in the progression of these disorders is still unknown. This fibrosis is characterized by stromal expansion and the excessive deposition of extracellular matrix (ECM) that replaces pancreatic tissue. This eventually leads to dysregulation of ECM turnover, production of cytokines, restriction of blood flow, and often exocrine and endocrine insufficiencies. Activated pancreatic stellate cells (PSCs) have been identified as key mediators in the progression of pancreatic fibrosis, serving as the predominant source of excess ECM proteins. Previously, we found that overexpression of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF) in pancreatic islets led to intraislet fibrosis. HB-EGF binds to and activates two receptors, epidermal growth factor receptor (EGFR) and ErbB4, as well as heparin moieties and CD9/DRAP27. To understand the mechanism underlying the induction of fibrogenesis by HB-EGF, we utilized a hypomorphic allele of Egfr, the Waved-2 allele, to demonstrate that EGFR signaling regulates fibrogenesis in vivo. Using an in vitro cell migration assay, we show that HB-EGF regulates both chemoattraction and stimulation of proliferation of PSCs via EGFR activation.

BMP4 is a novel paracrine inhibitor of liver regeneration

Transforming growth factor (TGF)-β family members exert strong effects on restoration of liver mass after injury. Bone morphogenetic proteins (BMPs) are members of the TGF-β family and are found in the liver, suggesting that these proteins may play a role in liver regeneration. We examined BMP signaling in the liver during hepatectomy. We found that BMP4 is constitutively expressed in the peribiliary stroma and endothelial cells of the liver and that expression is decreased after hepatectomy. Mice driven to maintain BMP4 expression in the liver display inhibited hepatocyte proliferation and restoration of liver mass after hepatectomy, suggesting that reduced BMP4 is necessary for normal regeneration. Consistent with this finding, hepatocyte-specific deletion of the BMP receptor activin receptor-like kinase 3 (Alk3) enhances regeneration and reduces phosphorylation of SMAD1/5/8, a transducer of BMP signaling. In contrast to experiments in wild-type mice, maintaining BMP4 levels has no effect on liver regeneration in hepatocyte-specific Alk3 null mice, providing evidence that BMP4 signals through Alk3 to inhibit liver regeneration. Consistent with these findings, the BMP4 antagonist Noggin enhances regeneration. Furthermore, high-dose BMP4 inhibits proliferation of primary hepatocytes and HepG2 cells in culture. These findings elucidate a new, potentially clinically relevant paradigm in which a constitutively expressed paracrine inhibitory factor plays a critical role in liver regeneration.

Heparin-binding epidermal growth factor-like growth factor eliminates constraints on activated Kras to promote rapid onset of pancreatic neoplasia

Pancreatic cancer remains as one of the most deadly cancers with few treatment options at late stages and little information about how it develops through earlier stages. Activating mutation of the Kras gene has been implicated in, but is not sufficient for, tumorigenesis. In mouse models of pancreatic cancer, loss of tumor suppressor genes in conjunction with Kras mutation leads to gradual stochastic acquisition of neoplastic precursors and carcinomas, whereas many cells remain phenotypically unaltered in younger mice. Here, we demonstrate that two oncogenic events, mutation of Kras and production of the growth factor heparin-binding epidermal growth factor-like growth factor (HB-EGF), are sufficient for rapid and complete neoplastic transformation of the exocrine pancreas. We found that macrophages are the major source of HB-EGF production in pancreatic cancer tissue samples, and that macrophages are present in high density and in close association with human pancreatic cancer lesions. In a mouse model, high macrophage density was observed at the earliest stages of neoplastic transformation. The consequence of elevated HB-EGF signaling was investigated without the confounding effects of other macrophage-produced factors via transgenic overexpression of the active form of HB-EGF. In this model, HB-EGF was sufficient to promote Kras-initiated tumorigenesis, inducing rapid and complete neoplastic transformation of the entire exocrine pancreas shortly after birth. HB-EGF overexpression and KrasG12D together, but neither alone, increased proliferation with increased cyclinD1 and decreased Cdkn2a/2d (p16/p19Ink4A/Arf). These findings establish the importance of oncogenic synergy in cancer initiation and promotion, and establish a molecular link between inflammation and the earliest stages of tumor induction.

Specific Activin Receptor-Like Kinase 3 Inhibitors Enhance Liver Regeneration

Pharmacologic agents to enhance liver regeneration after injury would have wide therapeutic application. Based on previous work suggesting inhibition of bone morphogenetic protein (BMP) signaling stimulates liver regeneration, we tested known and novel BMP inhibitors for their ability to accelerate regeneration in a partial hepatectomy (PH) model. Compounds were produced based on the 3,6-disubstituted pyrazolo[1,5-a] pyrimidine core of the BMP antagonist dorsomorphin and evaluated for their ability to inhibit BMP signaling and enhance liver regeneration. Antagonists of the BMP receptor activin receptor–like kinase 3 (ALK3), including LDN-193189 (LDN; 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), DMH2 (4-(2-(4-(3-(quinolin-4-yl)pyrazolo[1,5-a]pyrimidin-6-yl)phenoxy)ethyl)morpholine; VU0364849), and the novel compound VU0465350 (7-(4-isopropoxyphenyl)-3-(1H-pyrazol-4-yl)imidazo[1,2-a]pyridine; VU5350), blocked SMAD phosphorylation in vitro and in vivo, and enhanced liver regeneration after PH. In contrast, an antagonist of the BMP receptor ALK2, VU0469381 (5-(6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinolone; 1LWY), did not affect liver regeneration. LDN did not affect liver synthetic or metabolic function. Mechanistically, LDN increased serum interleukin-6 levels and signal transducer and activator of transcription 3 phosphorylation in the liver, and modulated other factors known to be important for liver regeneration, including suppressor of cytokine signaling 3 and p53. These findings suggest that inhibition of ALK3 may be part of a therapeutic strategy for treating human liver disease.

Transmembrane and Soluble Isoforms of Heparin-Binding Epidermal Growth Factor–Like Growth Factor Regulate Distinct Processes in the Pancreas

BACKGROUND & AIMS Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is produced as a type-I, single-pass transmembrane protein that can be cleaved to release a diffusible peptide. HB-EGF, often overexpressed in damaged or diseased epithelium, is normally expressed in pancreatic islets, but its function is not understood. METHODS To understand the function of each isoform of HB-EGF, we made transgenes expressing either a constitutively transmembrane or a constitutively secreted protein. RESULTS The transmembrane isoform was not an inert precursor protein, but a functional molecule, downregulating the glucose-sensing apparatus of pancreatic islets. Conversely, the secreted form of HB-EGF improved islet function, but had severe fibrotic and neoplastic effects on surrounding tissues. Each isoform had a more severe phenotype than that of full-length HB-EGF, even though the full-length protein was efficiently cleaved, thus producing both isoforms, suggesting that a level of regulation was lost by separating the isoforms. CONCLUSIONS This work demonstrates that islet function depends on the ratio of cleaved to uncleaved HB-EGF and that the transmembrane intermediate, while deleterious to islet function, is necessary to restrict action of soluble HB-EGF away from surrounding tissue.

SOCS2 Balances Metabolic and Restorative Requirements During Liver Regeneration

After significant injury, the liver must maintain homeostasis during the regenerative process. We hypothesized the existence of mechanisms to limit hepatocyte proliferation after injury to maintain metabolic and synthetic function. A screen for candidates revealed suppressor of cytokine signaling 2 (SOCS2), an inhibitor of growth hormone (GH) signaling, was strongly induced after partial hepatectomy. Using genetic deletion and administration of various factors we investigated the role of SOCS2 during liver regeneration. SOCS2 preserves liver function by restraining the first round of hepatocyte proliferation after partial hepatectomy by preventing increases in growth hormone receptor (GHR) via ubiquitination, suppressing GH pathway activity. At later times, SOCS2 enhances hepatocyte proliferation by modulating a decrease in serum insulin-like growth factor 1 (IGF-1) that allows GH release from the pituitary. SOCS2, therefore, plays a dual role in modulating the rate of hepatocyte proliferation. In particular, this is the first demonstration of an endogenous mechanism to limit hepatocyte proliferation after injury.

Dicer-Dependent Production of MicroRNA221 in Hepatocytes Inhibits p27 and is Required for Liver Regeneration in Mice.

Dicer processes microRNAs (miRs) into active forms in a wide variety of tissues, including the liver. To determine the role of Dicer in liver regeneration, we performed a series of in vivo and in vitro studies in a murine 2/3 hepatectomy model. Dicer was downregulated after 2/3 hepatectomy, and loss of Dicer inhibited liver regeneration associated with decreased cyclin A2 and miR-221, as well as increased levels of the cell cycle inhibitor p27. In vitro, miR-221 inhibited p27 production in primary hepatocytes and increased hepatocyte proliferation. Specific reconstitution of miR-221 in hepatocyte-specific Dicer-null mice inhibited p27 and restored liver regeneration. In wild type mice, targeted inhibition of miR-221 using a cholesterol-conjugated miR-221 inhibited hepatocyte proliferation after 2/3 hepatectomy. These results identify Dicer production of miR-221 as an essential component of a miRNA-dependent mechanism for suppression of p27 that controls the rate of hepatocyte proliferation after partial hepatectomy.NEW & NOTEWORTHY Our findings demonstrate a direct role for microRNAs in controlling the rate of liver regeneration after injury. By deleting Dicer, an enzyme responsible for processing microRNAs into mature forms, we determined miR-221 is a critical microRNA in the physiological process of restoration of liver mass after injury. miR-221 suppresses p27, releasing its inhibitory effects on hepatocyte proliferation. Pharmaceuticals based on miR-221 may be useful to modulate hepatocyte proliferation in the setting of liver injury.