Christine P. Petersen

Connective tissue growth factor acts within both endothelial cells and β cells to promote proliferation of developing β cells

Type 1 and type 2 diabetes result from an absolute or relative reduction in functional β-cell mass. One approach to replacing lost β-cell mass is transplantation of cadaveric islets; however, this approach is limited by lack of adequate donor tissue. Therefore, there is much interest in identifying factors that enhance β-cell differentiation and proliferation in vivo or in vitro. Connective tissue growth factor (CTGF) is a secreted molecule expressed in endothelial cells, pancreatic ducts, and embryonic β cells that we previously showed is required for β-cell proliferation, differentiation, and islet morphogenesis during development. The current study investigated the tissue interactions by which CTGF promotes normal pancreatic islet development. We found that loss of CTGF from either endothelial cells or β cells results in decreased embryonic β-cell proliferation, making CTGF unique as an identified β cell-derived factor that regulates embryonic β-cell proliferation. Endothelial CTGF inactivation was associated with decreased islet vascularity, highlighting the proposed role of endothelial cells in β-cell proliferation. Furthermore, CTGF overexpression in β cells during embryogenesis using an inducible transgenic system increased islet mass at birth by promoting proliferation of immature β cells, in the absence of changes in islet vascularity. Together, these findings demonstrate that CTGF acts in an autocrine manner during pancreas development and suggest that CTGF has the potential to enhance expansion of immature β cells in directed differentiation or regeneration protocols.

Macrophages Promote Progression of Spasmolytic Polypeptide-Expressing Metaplasia After Acute Loss of Parietal Cells

BACKGROUND & AIMS Loss of parietal cells causes the development of spasmolytic polypeptide-expressing metaplasia (SPEM) through transdifferentiation of chief cells. In the presence of inflammation, SPEM can advance into a more proliferative metaplasia with increased expression of intestine-specific transcripts. We used L635 to induce acute SPEM with inflammation in mice and investigated the roles of inflammatory cells in the development of SPEM. METHODS To study the adaptive immune system, Rag1 knockout, interferon-γ-deficient, and wild-type (control) mice received L635 for 3 days. To study the innate immune system, macrophages were depleted by intraperitoneal injection of clodronate liposomes 2 days before and throughout L635 administration. Neutrophils were depleted by intraperitoneal injection of an antibody against Ly6G 2 days before and throughout L635 administration. Pathology and immunohistochemical analyses were used to determine depletion efficiency, metaplasia, and proliferation. To characterize SPEM in each model, gastric tissues were collected and levels of Cftr, Dmbt1, and Gpx2 mRNAs were measured. Markers of macrophage polarization were used to identify subpopulations of macrophages recruited to the gastric mucosa. RESULTS Administration of L635 to Rag1 knockout, interferon-γ-deficient, and neutrophil-depleted mice led to development of proliferative SPEM and up-regulation of intestine-specific transcripts in SPEM cells, similar to controls. However, macrophage-depleted mice given L635 showed significant reductions in numbers of SPEM cells, SPEM cell proliferation, and expression of intestine-specific transcripts, compared with control mice given L635. In mice given L635, as well as patients with intestinal metaplasia, M2 macrophages were the primary inflammatory component. CONCLUSIONS Results from studies of mouse models and human metaplastic tissues indicate that M2 macrophages promote the advancement of SPEM in the presence of inflammation.

miR-30-HNF4γ and miR-194-NR2F2 regulatory networks contribute to the upregulation of metaplasia markers in the stomach

Objective Intestinal metaplasia and spasmolytic polypeptide-expressing metaplasia (SPEM) are considered neoplastic precursors of gastric adenocarcinoma and are both marked by gene expression alterations in comparison to normal stomach. Since miRNAs are important regulators of gene expression, we sought to investigate the role of miRNAs on the development of stomach metaplasias. Design We performed miRNA profiling using a quantitative reverse transcription-PCR approach on laser capture microdissected human intestinal metaplasia and SPEM. Data integration of the miRNA profile with a previous mRNA profile from the same samples was performed to detect potential miRNA-mRNA regulatory circuits. Transfection of gastric cancer cell lines with selected miRNA mimics and inhibitors was used to evaluate their effects on the expression of putative targets and additional metaplasia markers. Results We identified several genes as potential targets of miRNAs altered during metaplasia progression. We showed evidence that HNF4γ (upregulated in intestinal metaplasia) is targeted by miR-30 and that miR-194 targets a known co-regulator of HNF4 activity, NR2F2 (downregulated in intestinal metaplasia). Intestinal metaplasia markers such as VIL1, TFF2 and TFF3 were downregulated after overexpression of miR-30a in a HNF4γ-dependent manner. In addition, overexpression of HNF4γ was sufficient to induce the expression of VIL1 and this effect was potentiated by downregulation of NR2F2. Conclusions The interplay of the two transcription factors HNF4γ and NR2F2 and their coordinate regulation by miR-30 and miR-194, respectively, represent a miRNA to transcription factor network responsible for the expression of intestinal transcripts in stomach cell lineages during the development of intestinal metaplasia.

Characterization of progressive metaplasia in the gastric corpus mucosa of Mongolian gerbils infected with Helicobacter pylori.

Spasmolytic polypeptide‐expressing metaplasia (SPEM) and intestinal metaplasia are considered neoplastic precursors of gastric adenocarcinoma in humans. Loss of parietal cells causes the development of SPEM in the gastric corpus and then chronic inflammation drives SPEM toward a more proliferative lineage. Mongolian gerbils infected with Helicobacter pylori develop chronic gastritis and metaplasia, mimicking aspects of human gastritis with H. pylori infection. We therefore examined metaplastic lineages in the gastric corpus mucosa of gerbils infected by H. pylori strain 7.13, which produces rapid onset of severe inflammation. Six weeks following H. pylori infection, Griffonia simplicifolia lectin II (GSII)‐positive SPEM developed in the base of oxyntic glands in association with parietal cell loss and inflammation. In association with severe inflammation, SPEM glands evolved into aberrant phenotypes, including branched lesions, dilated lesions, and penetrating invasive glands. Mucin 4 (MUC4) was up‐regulated in SPEM and progressive SPEM. Clusterin was expressed in the tips of branched and dilated lesions and throughout regions of invasive glands. Intriguingly, clusterin‐positive regions in these lesions expressed Ki67 and matrix metalloproteinase 7 (MMP‐7). These same regions were also positive for expression of phospho‐IkBα, suggestive of activated NFkB signalling. These findings suggest that clusterin‐positive regions in progressive phenotypes of SPEM have invasive characteristics. Thus, H. pylori infection in gerbils induces SPEM, which then can progress to further aberrant and invasive metaplastic phenotypes. Copyright

Murine models of gastric corpus preneoplasia

Intestinal-type gastric adenocarcinoma evolves in a field of pre-existing metaplasia. Over the past 20 years, a number of murine models have been developed to address aspects of the physiology and pathophysiology of metaplasia induction. Although none of these models has achieved true recapitulation of the induction of adenocarcinoma, they have led to important insights into the factors that influence the induction and progression of metaplasia. Here, we review the pathologic definitions relevant to alterations in gastric corpus lineages and classification of metaplasia by specific lineage markers. In addition, we review present murine models of the induction and progression of spasmolytic polypeptide (TFF2)–expressing metaplasia, the predominant metaplastic lineage observed in murine models. These models provide a basis for the development of a broader understanding of the physiological and pathophysiological roles of metaplasia in the stomach.

Loss of HNF6 expression correlates with human pancreatic cancer progression

Normal pancreatic epithelium progresses through various stages of pancreatic intraepithelial neoplasms (PanINs) in the development of pancreatic ductal adenocarcinoma (PDAC). Transcriptional regulation of this progression is poorly understood. In mouse, the hepatic nuclear factor 6 (Hnf6) transcription factor is expressed in ductal cells and at lower levels in acinar cells of the adult pancreas, but not in mature endocrine cells. Hnf6 is critical for terminal differentiation of the ductal epithelium during embryonic development and for pancreatic endocrine cell specification. We previously showed that, in mice, loss of Hnf6 from the pancreatic epithelium during organogenesis results in increased duct proliferation and altered duct architecture, increased periductal fibrosis and acinar-to-ductal metaplasia. Here we show that decreased expression of HNF6 is strongly correlated with increased severity of PanIN lesions in samples of human pancreata and is absent from >90% of PDAC. Mouse models in which cancer progression can be analyzed from the earliest stages that are seldom accessible in humans support a role for Hnf6 loss in progression from early- to late-stage PanIN and PDAC. In addition, gene expression analyses of human pancreatic cancer reveal decreased expression of HNF6 and its direct and indirect target genes compared with normal tissue and upregulation of genes that act in opposition to HNF6 and its targets. The negative correlation between HNF6 expression and pancreatic cancer progression suggests that HNF6 maintains pancreatic epithelial homeostasis in humans, and that its loss contributes to the progression from PanIN to ductal adenocarcinoma. Insight on the role of HNF6 in pancreatic cancer development could lead to its use as a biomarker for early detection and prognosis.

Dynamic Expansion of Gastric Mucosal Doublecortin-Like Kinase 1–Expressing Cells in Response to Parietal Cell Loss Is Regulated by Gastrin

Doublecortin-like kinase 1 (Dclk1) is considered a reliable marker for tuft cells in the gastrointestinal tract. We investigated the dynamic changes of tuft cells associated with mouse models of oxyntic atrophy and metaplasia in the stomach. Increases in the numbers of Dclk1-positive tuft cells were observed in several models of parietal cell loss. However, the expanded population of Dclk1-expressing cells showed a morphologically distinct structure in apical microvilli and acetylated microtubules, which was not seen in the tuft cells present in the normal gastric mucosa. These microvillar sensory cells (MVSCs) showed no evidence of proliferation. The expansion of the MVSCs induced by oxyntic atrophy was reversible after the return of parietal cells. More important, expansion of MVSCs after induced parietal cell loss was not observed in Gast(-/-) mice. Although the Dclk1-expressing cells in the normal gastric mucosa were in part derived from Lrig1-expressing stem cells, the Lrig1-lineaged cells did not produce the expanded Dclk1-expressing cells associated with oxyntic atrophy. These studies indicate that loss of parietal cells leads to the reversible emergence of a novel Dclk1-expressing sensory cell population in the gastric mucosa.

Maturity and age influence chief cell ability to transdifferentiate into metaplasia

The plasticity of gastric chief cells is exemplified by their ability to transdifferentiate into spasmolytic polypeptide-expressing metaplasia (SPEM) after parietal cell loss. We sought to determine if chief cell maturity is a limiting factor in the capacity to transdifferentiate. Mist1-/- mice, previously shown to form only immature chief cells, were treated with DMP-777 or L635 to study the capability of these immature chief cells to transdifferentiate into a proliferative metaplastic lineage after acute parietal cell loss. Mist1-/- mice treated with DMP-777 showed fewer chief cell to SPEM transitions. Mist1-/- mice treated with L635 demonstrated significantly fewer proliferative SPEM cells compared with control mice. Thus immature chief cells were unable to transdifferentiate efficiently into SPEM after acute parietal cell loss. To determine whether chief cell age affects transdifferentiation into SPEM, we used tamoxifen to induce YFP expression in chief cells of Mist1CreER/+;RosaYFP mice and subsequently treated the cells with L635 to induce SPEM at 1 to 3.5 mo after tamoxifen treatment. After L635 treatment to induce acute parietal cell loss, 43% of all YFP-positive cells at 1 mo posttamoxifen were SPEM cells, of which 44% of these YFP-positive SPEM cells were proliferative. By 2 mo after tamoxifen induction, only 24% of marked SPEM cells were proliferating. However, by 3.5 mo after tamoxifen induction, only 12% of marked chief cells transdifferentiated into SPEM and none were proliferative. Thus, as chief cells age, they lose their ability to transdifferentiate into SPEM and proliferate. Therefore, both functional maturation and age limit chief cell plasticity. NEW & NOTEWORTHY Previous investigations have indicated that spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach arises from transdifferentiation of chief cells. Nevertheless, the intrinsic properties of chief cells that influence transdifferentiation have been largely unknown. We now report that the ability to transdifferentiate into SPEM is impaired in chief cells that lack full functional maturation, and as chief cells age, they lose their ability to transdifferentiate. Thus chief cell plasticity is dependent on both cell age and maturation.

Establishment of novel in vitro mouse chief cell and SPEM cultures identifies MAL2 as a marker of metaplasia in the stomach

Oxyntic atrophy in the stomach leads to chief cell transdifferentiation into spasmolytic polypeptide expressing metaplasia (SPEM). Investigations of preneoplastic metaplasias in the stomach are limited by the sole reliance on in vivo mouse models, owing to the lack of in vitro models for distinct normal mucosal lineages and metaplasias. Utilizing the Immortomouse, in vitro cell models of chief cells and SPEM were developed to study the characteristics of normal chief cells and metaplasia. Chief cells and SPEM cells isolated from Immortomice were cultured and characterized at both the permissive (33°C) and the nonpermissive temperature (39°C). Clones were selected on the basis of their transcriptional expression of specific stomach lineage markers (named ImChief and ImSPEM) and protein expression and growth were analyzed. The transcriptional expression profiles of ImChief and ImSPEM cells were compared further by using gene microarrays. ImChief cells transcriptionally express most chief cell markers and contain pepsinogen C and RAB3D-immunostaining vesicles. ImSPEM cells express the SPEM markers TFF2 and HE4 and constitutively secrete HE4. Whereas ImChief cells cease proliferation at the nonpermissive temperature, ImSPEM cells continue to proliferate at 39°C. Gene expression profiling of ImChief and ImSPEM revealed myelin and lymphocyte protein 2 (MAL2) as a novel marker of SPEM lineages. Our results indicate that the expression and proliferation profiles of the novel ImChief and ImSPEM cell lines resemble in vivo chief and SPEM cell lineages. These cell culture lines provide the first in vitro systems for studying the molecular mechanisms of the metaplastic transition in the stomach.

A signalling cascade of IL-33 to IL-13 regulates metaplasia in the mouse stomach

Objective Alternatively activated macrophages (M2) are associated with the progression of spasmolytic polypeptide-expressing metaplasia (SPEM) in the stomach. However, the precise mechanism(s) and critical mediators that induce SPEM are unknown. Design To determine candidate genes important in these processes, macrophages from the stomach corpus of mice with SPEM (DMP-777-treated) or advanced SPEM (L635-treated) were isolated and RNA sequenced. Effects on metaplasia development after acute parietal cell loss induced by L635 were evaluated in interleukin (IL)-33, IL-33 receptor (ST2) and IL-13 knockout (KO) mice. Results Profiling of metaplasia-associated macrophages in the stomach identified an M2a-polarised macrophage population. Expression of IL-33 was significantly upregulated in macrophages associated with advanced SPEM. L635 induced metaplasia in the stomachs of wild-type mice, but not in the stomachs of IL-33 and ST2 KO mice. While IL-5 and IL-9 were not required for metaplasia induction, IL-13 KO mice did not develop metaplasia in response to L635. Administration of IL-13 to ST2 KO mice re-established the induction of metaplasia following acute parietal cell loss. Conclusions Metaplasia induction and macrophage polarisation after parietal cell loss is coordinated through a cytokine signalling network of IL-33 and IL-13, linking a combined response to injury by both intrinsic mucosal mechanisms and infiltrating M2 macrophages.