Hepatic stellate cells maintain liver homeostasis through paracrine neurotrophin-3 signaling that induces hepatocyte proliferation.

Organ size is maintained by the controlled proliferation of distinct cell populations. In the mouse liver, hepatocytes in the midlobular zone that are positive for cyclin D1 (CCND1) repopulate the parenchyma at a constant rate to preserve liver mass. Here, we investigated how hepatocyte proliferation is supported by hepatic stellate cells (HSCs), pericytes that are in close proximity to hepatocytes. We used T cells to ablate nearly all HSCs in the murine liver, enabling the unbiased characterization of HSC functions. In the normal liver, complete loss of HSCs persisted for up to 10 weeks and caused a gradual reduction in liver mass and in the number of CCND1+ hepatocytes. We identified neurotrophin-3 (Ntf-3) as an HSC-produced factor that induced the proliferation of midlobular hepatocytes through the activation of tropomyosin receptor kinase B (TrkB). Treating HSC-depleted mice with Ntf-3 restored CCND1+ hepatocytes in the midlobular region and increased liver mass. These findings establish that HSCs form the mitogenic niche for midlobular hepatocytes and identify Ntf-3 as a hepatocyte growth factor.

Supermeres are functional extracellular nanoparticles replete with disease biomarkers and therapeutic targets.

Extracellular vesicles and exomere nanoparticles are under intense investigation as sources of clinically relevant cargo. Here we report the discovery of a distinct extracellular nanoparticle, termed supermere. Supermeres are morphologically distinct from exomeres and display a markedly greater uptake in vivo compared with small extracellular vesicles and exomeres. The protein and RNA composition of supermeres differs from small extracellular vesicles and exomeres. Supermeres are highly enriched with cargo involved in multiple cancers (glycolytic enzymes, TGFBI, miR-1246, MET, GPC1 and AGO2), Alzheimer's disease (APP) and cardiovascular disease (ACE2, ACE and PCSK9). The majority of extracellular RNA is associated with supermeres rather than small extracellular vesicles and exomeres. Cancer-derived supermeres increase lactate secretion, transfer cetuximab resistance and decrease hepatic lipids and glycogen in vivo. This study identifies a distinct functional nanoparticle replete with potential circulating biomarkers and therapeutic targets for a host of human diseases.

Integrin ß1 Establishes Liver Microstructure and Modulates Transforming Growth Factor ß during Liver Development and Regeneration.

A unique and complex microstructure underlies the diverse functions of the liver. Breakdown of this organization, as occurs in fibrosis and cirrhosis, impairs liver function and leads to disease. The role of integrin ß1 was examined both in establishing liver microstructure and recreating it after injury. Embryonic deletion of integrin ß1 in the liver disrupts the normal development of hepatocyte polarity, specification of cell-cell junctions, and canalicular formation. This in turn leads to the expression of transforming growth factor ß (TGF-ß) and widespread fibrosis. Targeted deletion of integrin ß1 in adult hepatocytes prevents recreation of normal hepatocyte architecture after liver injury, with resultant fibrosis. In vitro, integrin ß1 is essential for canalicular formation and is needed to prevent stellate cell activation by modulating TGF-ß. Taken together, these findings identify integrin ß1 as a key determinant of liver architecture with a critical role as a regulator of TGF-ß secretion. These results suggest that disrupting the hepatocyte-extracellular matrix interaction is sufficient to drive fibrosis.

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.

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.

Epithelial tissues have varying degrees of susceptibility to Kras(G12D)-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, Kras(G12D), 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 Kras(G12D) 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 Kras(G12D)-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 TGFalpha, 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.

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.

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.

A CK19(CreERT) 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 K19(CreERT) mouse line in which the tamoxifen-activable CreER(T) 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.

Overexpression of heparin-binding EGF-like growth factor in mouse pancreas results in fibrosis and epithelial metaplasia.

BACKGROUND & AIMS: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is expressed in both normal pancreatic islets and in pancreatic cancers, but its role in pancreatic physiology and disease is not known. This report examines the effects of HB-EGF overexpression in mouse pancreas. METHODS: Transgenic mice were established using a tissue-specific promoter to express an HB-EGF complementary DNA in pancreatic beta cells, effectively elevating HB-EGF protein 3-fold over endogenous levels. RESULTS: Mice overexpressing HB-EGF in pancreatic islets showed both endocrine and exocrine pancreatic defects. Initially, islets from transgenic mice failed to segregate alpha, beta, delta, and PP cells appropriately within islets, and had impaired separation from ducts and acini. Increased stroma was detected within transgenic islets, expanding with age to cause fibrosis of both endocrine and exocrine compartments. In addition to these structural abnormalities, subsets of transgenic mice developed profound hyperglycemia and/or proliferation of metaplastic ductal epithelium. Both conditions were associated with severe stromal expansion, suggesting a role for islet/stromal interaction in the onset of the pancreatic disease initiated by HB-EGF. Supporting this conclusion, primary mouse fibroblasts adhered to transgenic islets when the 2 tissues were cocultured in vitro, but did not interact with nontransgenic islets. CONCLUSIONS: An elevation in HB-EGF protein in pancreatic islets led to altered interactions among islet cells and among islets, stromal tissues, and ductal epithelium. Many of the observed phenotypes appeared to involve altered cell adhesion. These data support a role for islet factors in the development of both endocrine and exocrine disease.