Re-expression of IGF-II is important for beta cell regeneration in adult mice.

BACKGROUND: The key factors which support re-expansion of beta cell numbers after injury are largely unknown. Insulin-like growth factor II (IGF-II) plays a critical role in supporting cell division and differentiation during ontogeny but its role in the adult is not known. In this study we investigated the effect of IGF-II on beta cell regeneration. METHODOLOGY/PRINCIPAL FINDINGS: We employed an in vivo model of 'switchable' c-Myc-induced beta cell ablation, pIns-c-MycER(TAM), in which 90% of beta cells are lost following 11 days of c-Myc (Myc) activation in vivo. Importantly, such ablation is normally followed by beta cell regeneration once Myc is deactivated, enabling functional studies of beta cell regeneration in vivo. IGF-II was shown to be re-expressed in the adult pancreas of pIns-c-MycER(TAM)/IGF-II(+/+) (MIG) mice, following beta cell injury. As expected in the presence of IGF-II beta cell mass and numbers recover rapidly after ablation. In contrast, in pIns-c-MycER(TAM)/IGF-II(+/-) (MIGKO) mice, which express no IGF-II, recovery of beta cell mass and numbers were delayed and impaired. Despite failure of beta cell number increase, MIGKO mice recovered from hyperglycaemia, although this was delayed. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that beta cell regeneration in adult mice depends on re-expression of IGF-II, and supports the utility of using such ablation-recovery models for identifying other potential factors critical for underpinning successful beta cell regeneration in vivo. The potential therapeutic benefits of manipulating the IGF-II signaling systems merit further exploration.

Myeloid, but not pancreatic, RelA/p65 is required for fibrosis in a mouse model of chronic pancreatitis.

BACKGROUND & AIMS: Little is known about how transcription factors might regulate pathogenesis of chronic pancreatitis (CP). We analyzed the in vivo role of RelA/p65, a component of the transcription factor nuclear factor (NF)-κB, in different cell types during development of CP in mice. METHODS: RelA/p65 was functionally inactivated in the pancreas (relaΔpanc), in myeloid cells (relaΔmye), or both (relaΔpanc,Δmye) compartments using the Cre-loxP strategy. Experimental CP was induced with repetitive injections of cerulein over 6 weeks. Pancreata were investigated histologically and biochemically. We created an in vitro coculture assay of pancreatic stellate cells (PSC) and macrophages and performed gene arrays from pancreata and macrophages with functionally inactivated RelA/p65. Tissue samples from patients with CP were analyzed for matrix metalloproteinase (MMP) 10 expression. RESULTS: In contrast to their relaF/F littermates, relaΔpanc displayed typical signs of CP after long-term stimulation with cerulein. Numerous macrophages and activated α-smooth muscle actin (SMA)-positive PSCs were detected. Additional inactivation of RelA/p65 in myeloid cells (relaΔpanc,Δmye) attenuated fibrosis. In vitro, RelA/p65-deficient, lipopolysaccharide (LPS)-stimulated macrophages degraded fibronectin in cocultured PSCs. Using gene expression analysis, MMP-10 was identified as a candidate for this process. Recombinant MMP-10 degraded fibronectin in LPS-stimulated PSCs. In tissue samples from patients with CP, MMP-10 was up-regulated in myeloid cells. CONCLUSIONS: RelA/p65 functions in myeloid cells to promote pathogenesis of CP. In acinar cells, RelA/p65 protects against chronic inflammation, whereas myeloid RelA/p65 promotes fibrogenesis. In macrophage, MMP-10 functions as a RelA/p65-dependent, potentially antifibrogenic factor during progression of CP.

Identification of epidermal Pdx1 expression discloses different roles of Notch1 and Notch2 in murine Kras(G12D)-induced skin carcinogenesis in vivo.

BACKGROUND: The Ras and Notch signaling pathways are frequently activated during development to control many diverse cellular processes and are often dysregulated during tumorigenesis. To study the role of Notch and oncogenic Kras signaling in a progenitor cell population, Pdx1-Cre mice were utilized to generate conditional oncogenic Kras(G12D) mice with ablation of Notch1 and/or Notch2. METHODOLOGY/PRINCIPAL FINDINGS: Surprisingly, mice with activated Kras(G12D) and Notch1 but not Notch2 ablation developed skin papillomas progressing to squamous cell carcinoma providing evidence for Pdx1 expression in the skin. Immunostaining and lineage tracing experiments indicate that PDX1 is present predominantly in the suprabasal layers of the epidermis and rarely in the basal layer. Further analysis of keratinocytes in vitro revealed differentiation-dependent expression of PDX1 in terminally differentiated keratinocytes. PDX1 expression was also increased during wound healing. Further analysis revealed that loss of Notch1 but not Notch2 is critical for skin tumor development. Reasons for this include distinct Notch expression with Notch1 in all layers and Notch2 in the suprabasal layer as well as distinctive p21 and ß-catenin signaling inhibition capabilities. CONCLUSIONS/SIGNIFICANCE: Our results provide strong evidence for epidermal expression of Pdx1 as of yet not identified function. In addition, this finding may be relevant for research using Pdx1-Cre transgenic strains. Additionally, our study confirms distinctive expression and functions of Notch1 and Notch2 in the skin supporting the importance of careful dissection of the contribution of individual Notch receptors.

Notch2 is required for progression of pancreatic intraepithelial neoplasia and development of pancreatic ductal adenocarcinoma.

Pancreatic cancer is one of the most fatal malignancies lacking effective therapies. Notch signaling is a key regulator of cell fate specification and pancreatic cancer development; however, the role of individual Notch receptors and downstream signaling is largely unknown. Here, we show that Notch2 is predominantly expressed in ductal cells and pancreatic intraepithelial neoplasia (PanIN) lesions. Using genetically engineered mice, we demonstrate the effect of conditional Notch receptor ablation in KrasG12D-driven pancreatic carcinogenesis. Deficiency of Notch2 but not Notch1 stops PanIN progression, prolongs survival, and leads to a phenotypical switch toward anaplastic pancreatic cancer with epithelial-mesenchymal transition. By expression profiling, we identified increased Myc signaling regulated by Notch2 during tumor development, placing Notch2 as a central regulator of PanIN progression and malignant transformation. Our study supports the concept of distinctive roles of individual Notch receptors in cancer development.

Conditional inactivation of Myc impairs development of the exocrine pancreas.

Recent studies have shown that Wnt/beta-catenin signaling is essential for development of the exocrine pancreas, but the role of beta-catenin-dependent target genes such as Myc during pancreatic development is not well known. Here, we show that tissue-specific deletion of Myc causes a slightly accelerated differentiation of pancreatic epithelial cells into endocrine cells and perturbs the proliferation of pancreatic progenitors and acinar precursor cells during early development, resulting in a severe reduction of the epithelial cell mass of pancreatic buds and an extensive acinar hypoplasia. Loss of Myc does not affect the expression of the tissue-specific transcription factor PTF1a, which is required for the differentiation of acinar cells. In contrast to its role for exocrine cell growth, the development of endocrine cell lineages is not significantly disturbed. These data suggest that Myc is required for the expansion of the exocrine pancreas. Our observations are consistent with the findings in beta-catenin-deficient pancreas, suggesting that Wnt/beta-catenin signaling affects the proliferation of pancreatic epithelial cells and acinar precursors through its target gene Myc.

A Cre-loxP-based mouse model for conditional somatic gene expression and knockdown in vivo by using avian retroviral vectors.

Site- and time-specific somatic gene transfer by using the avian sarcoma-leukosis retrovirus RCAS (replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor) has been shown to be a powerful tool to analyze gene function in vivo. RCAS retroviruses that express the avian subgroup A envelope transduce only mammalian cells genetically engineered to express the avian retroviral receptor, tumor virus A (TVA). Here, we generated a knockin mouse line termed LSL-R26(Tva-lacZ) with concomitant conditional expression of TVA and lacZ by targeting the Rosa26 locus. A loxP-flanked transcriptional stop cassette was used for conditional activation of TVA and LacZ expression in a Cre-recombinase-dependent manner. To demonstrate the ability of this system for conditional somatic gene transfer in vivo, we directed TVA expression to the pancreas. Introduction of an RCAS vector with Bryan-RSV polymerase and subgroup A envelope [RCASBP(A)] carrying oncogenic Kras(G12D) induced focal ductal pancreatic lesions that recapitulate human pancreatic intraepithelial neoplasias that progress to pancreatic ductal adenocarcinomas. TVA-mediated infection of genetically engineered mice with endogenous expression of Kras(G12D) in pancreatic progenitor cells by using RCASBP(A) virus carrying a short hairpin RNA directed against murine TP53, resulted in dramatically enhanced progression to invasive adenocarcinomas. These results show that conditional expression of TVA enables spatiotemporal gene expression and knockdown in a small subset of somatic cells in vivo. Therefore, it closely models carcinogenesis in humans where tumors evolve from somatic gene mutations in developmentally normal cells. Combined with the growing number of Cre expression models, RCAS-TVA-based gene expression and knockdown systems open up promising perspectives for analysis of gene function in a time-controlled and tissue-specific fashion in vitro and in vivo.

Conditional ablation of Notch signaling in pancreatic development.

The role of the Notch signaling members Notch1, Notch2 and Rbpj in exocrine pancreatic development is not well defined. We therefore analyzed conditional pancreas-specific Rbpj and combined Notch1/Notch2 knockout mice using Ptf1a(+/Cre(ex1)) mice crossed with floxed Rbpj or Notch1/Notch2 mice. Mice were analyzed at different embryonic stages for pancreatic exocrine and endocrine development. The absence of Rbpj in pancreatic progenitor cells impaired exocrine pancreas development up to embryonic day 18.5 and led to premature differentiation of pancreatic progenitors into endocrine cells. In Rbpj-deficient pancreata, amylase-expressing acini and islets formed during late embryonic and postnatal development, suggesting an essential role of Rbpj in early but not late development. Contrary to this severe phenotype, the concomitant inactivation of Notch1 and Notch2 only moderately disturbed the proliferation of pancreatic epithelial cells during early embryonic development, and did not inhibit pancreatic development. Our results show that, in contrast to Rbpj, Notch1 and Notch2 are not essential for pancreatogenesis. These data favor a Notch-independent role of Rbpj in the development of the exocrine pancreas. Furthermore, our findings suggest that in late stages of pancreatic development exocrine cell differentiation and maintenance are independent of Rbpj.

Notch signaling is required for exocrine regeneration after acute pancreatitis.

BACKGROUND & AIMS: The mechanisms for tissue regeneration and renewal after acute pancreatitis are not well understood but may involve activation of Notch signaling. To study the effect of Notch signaling ablation during acute experimental pancreatitis, we used a chemical and genetic approach to ablate Notch signaling in cerulein-induced pancreatitis in mice. METHODS: Acute pancreatitis was induced by cerulein treatment in mice treated with the gamma-secretase inhibitor dibenzazepine or in conditional Notch1 knockout mice. Mice were characterized using immunohistologic, biochemical, and molecular methods. To investigate Notch and beta-catenin interaction, acinar 266-6 cells were analyzed using transfection and biochemical assays. RESULTS: Loss of Notch signaling results in impaired regeneration after acute pancreatitis with fewer mature acinar cells in dibenzazepine-treated and Notch1-deficient mice in the regenerative phase 3 days after induction. beta-catenin expression was increased and prolonged during exocrine regeneration. Crosstalk between Notch and beta-catenin-mediated signaling was identified, with Notch1-IC inhibiting beta-catenin-mediated transcriptional activity. This inhibition was dependent on a functional RAM domain. CONCLUSIONS: Inhibition of Notch signaling in vivo leads to impaired regeneration of the exocrine pancreas after acute pancreatitis. Our results suggest an interaction of Notch and Wnt signaling in pancreatic acinar cells, providing evidence for a role of these pathways in the regulation of the maturation process of acinar cells.

Pancreas-specific RelA/p65 truncation increases susceptibility of acini to inflammation-associated cell death following cerulein pancreatitis.

Activation of the transcription factor NF-kappaB/Rel has been shown to be involved in inflammatory disease. Here we studied the role of RelA/p65, the main transactivating subunit, during acute pancreatitis using a Cre-loxP strategy. Selective truncation of the rela gene in pancreatic exocrine cells led to both severe injury of the acinar cells and systemic complications including lung and liver damage. Our data demonstrated that expression and induction of the protective pancreas-specific acute phase protein pancreatitis-associated protein 1 (PAP1) depended on RelA/p65. Lentiviral gene transfer of PAP1 cDNA reduced the extent of necrosis and infiltration in the pancreata of mice with selective truncation of RelA/p65. These results provide in vivo evidence for RelA/p65 protection of acinar cell death via upregulation of PAP1. Moreover, our data underscore the pancreas-specific role of NF-kappaB/Rel and suggest multidimensional roles of NF-kappaB/Rel in different cells and contexts during inflammation.

Ptf1a is essential for the differentiation of GABAergic and glycinergic amacrine cells and horizontal cells in the mouse retina.

Basic helix-loop-helix (bHLH) transcription factors are important regulators of retinal neurogenesis. In the developing retina, proneural bHLH genes have highly defined expressions, which are influenced by pattern formation and cell-specification pathways. We report here that the tissue-specific bHLH transcription factor Ptf1a (also known as PTF1-p48) is expressed from embryonic day 12.5 of gestation (E12.5) to postnatal day 3 (P3) during retinogenesis in the mouse. Using recombination-based lineage tracing, we provide evidence that Ptf1a is expressed in precursors of amacrine and horizontal cells. Inactivation of Ptf1a in the developing retina led to differentiation arrest of amacrine and horizontal precursor cells in addition to partial transdifferentiation of Ptf1a-expressing precursor cells to ganglion cells. Analysis of late cell-type-specific markers revealed the presence of a small population of differentiated amacrine cells, whereas GABAergic and glycinergic amacrine cells, as well as horizontal cells, were completely missing in Ptf1a-knockout retinal explants. We conclude that Ptf1a contributes to the differentiation of horizontal cells and types of amacrine cells during mouse retinogenesis.