Robert H. Whitehead

Keratinocyte growth factor induces proliferation of hepatocytes and epithelial cells throughout the rat gastrointestinal tract.

Keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, was identified as a specific keratinocyte mitogen after isolation from a lung fibroblast line. Recently, recombinant (r)KGF was found to influence proliferation and differentiation patterns of multiple epithelial cell lineages within skin, lung, and the reproductive tract. In the present study, we designed experiments to identify additional target tissues, and focused on the rat gastrointestinal (GI) system, since a putative receptor, K-sam, was originally identified in a gastric carcinoma. Expression of KGF receptor and KGF mRNA was detected within the entire GI tract, suggesting the gut both synthesized and responded to KGF. Therefore, rKGF was administered to adult rats and was found to induce markedly increased proliferation of epithelial cells from the foregut to the colon, and of hepatocytes, one day after systemic treatment. Daily treatment resulted in the marked selective induction of mucin-producing cell lineages throughout the GI tract in a dose-dependent fashion. Other cell lineages were either unaffected (e.g., Paneth cells), or relatively decreased (e.g., parietal cells, enterocytes) in rKGF-treated rats. The direct effect of rKGF was confirmed by demonstrating markedly increased carcinoembryonic antigen production in a human colon carcinoma cell line, LIM1899. Serum levels of albumin were specifically and significantly elevated after daily treatment. These results demonstrate rKGF can induce epithelial cell activation throughout the GI tract and liver. Further, endogenous KGF may be a normal paracrine mediator of growth within the gut.

Stimulated Shedding of Vascular Cell Adhesion Molecule 1 (VCAM-1) Is Mediated by Tumor Necrosis Factor-α-converting Enzyme (ADAM 17)

A variety of cell surface adhesion molecules can exist as both transmembrane proteins and soluble circulating forms. Increases in the levels of soluble adhesion molecules have been correlated with a variety of inflammatory diseases, suggesting a pathological role. Although soluble forms are thought to result from proteolytic cleavage from the cell surface, relatively little is known about the proteases responsible for their release. In this report we demonstrate that under normal culture conditions, cells expressing vascular cell adhesion molecule 1 (VCAM-1) release a soluble form of the extracellular domain that is generated by metalloproteinase-mediated cleavage. VCAM-1 release can be rapidly simulated by phorbol 12-myristate 13-acetate (PMA), and this induced VCAM-1 shedding is mediated by metalloproteinase cleavage of VCAM-1 near the transmembrane domain. PMA-induced VCAM-1 shedding occurs as the result of activation of a specific pathway, as the generation of soluble forms of three other adhesion molecules, E-selectin, platelet-endothelial cell adhesion molecule 1, and intercellular adhesion molecule 1, are not altered by PMA stimulation. Using cells derived from genetically deficient mice, we identify tumor necrosis factor-α-converting enzyme (TACE or ADAM 17) as the protease responsible for PMA-induced VCAM-1 release, including shedding of endogenously expressed VCAM-1 by murine endothelial cells. Therefore, TACE-mediated shedding of VCAM-1 may be important for the regulation of VCAM-1 function at the cell surface.

Clonogenic Growth of Epithelial Cells From Normal Colonic Mucosa From Both Mice and Humans

BACKGROUND & AIMS The factors controlling the proliferation and differentiation of the colonic mucosa are unknown and have proved difficult to identify mainly because of a lack of in vitro methods for studying the proliferative cells of the mucosa. METHODS We have developed a novel method of preparing a viable single-cell suspension from isolated crypts and cloning these single cells. RESULTS We have obtained clonogenic growth from this single-cell suspension with an average of 1 colony per 10(5) cells in control cultures. Addition of conditioned medium from the LIM1863 colon carcinoma cell line increased the mean colony number to 11 +/- 3 per 10(5) cells. The cells forming the colonies are still viable after 4 weeks in culture. The epithelial nature of the cells was confirmed by ultrastructural and immunohistochemical methods with staining for keratin 8 and 18 and anti-human epithelial membrane-specific antigen and a positive result on polymerase chain reaction for keratin 19. CONCLUSIONS We have successfully cloned single cells from disaggregated colonic crypts from both human and murine colonic mucosa. We have also demonstrated the presence of an active clonogenic factor in the conditioned medium of a colon carcinoma cell line. Assays show that the clonogenic activity in the conditioned medium is not caused by the presence of any of the epidermal growth factor family of growth factors. This is the first report of a clonogenic assay for epithelial cells of normal colonic mucosa.

Synergy between Apc min and an activated ras mutation is sufficient to induce colon carcinomas.

Colon carcinomas appear to arise from the cumulative effect of mutations to several genes (APC, DCC, p53, ras, hMLH1, and hMSH2). By using novel colonic epithelial cell lines derived from the Immorto mouse, named the YAMC (young adult mouse colon) cell line, and an Immorto-Min mouse hybrid, named the IMCE (Immorto-Min colonic epithelial) cell line, carrying the Apc min mutation, we investigated the effect of an activated v-Ha-ras gene on tumor progression. The YAMC and IMCE cell lines are normal colonic epithelial cell lines which are conditionally immortalized by virtue of expression of a temperature-sensitive simian virus 40 (SV40) large T antigen. Under conditions which permit expression of a functional SV40 large T antigen (33 degrees C plus gamma interferon), neither the YAMC nor the IMCE cell line grows in soft agar or is tumorigenic in nude mice. In vitro, when the SV40 large T antigen is inactivated (39 degrees C without gamma interferon), the cells stop proliferating and die. By infecting the YAMC and IMCE cell lines with a replication-defective psi2-v-Ha-ras virus, we derived cell lines which overexpress the v-Ha-ras gene (YAMC-Ras and IMCE-Ras). In contrast to the parental cell lines, under conditions in which the SV40 large T antigen is inactive, both the YAMC-Ras and IMCE-Ras cell lines continue to proliferate. Initally YAMC-Ras cells do not form tumors; however, tumors are visible after 90 days of incubation. IMCE-Ras cells form colonies in soft agar under both permissive and nonpermissive culture conditions. Furthermore, IMCE-Ras cells form tumors in nude mice within 3 weeks. The phenotype of the IMCE-Ras cell line thus clearly demonstrates that a defective Apc allele and an activated ras gene are sufficient to transform normal colonic epithelial cells and render them tumorigenic.

Inactivation of TGF-beta signaling in hepatocytes results in an increased proliferative response after partial hepatectomy.

The transforming growth factor β (TGF-β) signaling pathway, which is activated by the TGF-β receptor complex consisting of type I and type II TGF-β receptors (TGFBR1 and TGFBR2), regulates cell growth and death. TGF-β and components of its signaling pathway, particularly TGFBR2, have been implicated as tumor suppressor genes and important antimitogenic factors in the gastrointestinal tract and liver. An in vivo approach to study these effects has been hindered by the embryonic lethality of Tgfbr2−/− mice and poor viability of the Tgfb1−/− mice. Consequently, we have developed a hepatocyte-specific Tgfbr2 knockout mouse, the Alb-cre Tgfbr2flx/flx mouse, to study the physiologically relevant effects of TGF-β signaling on epithelial cell proliferation in vivo. After 70% hepatectomy, we observed increased proliferation and an increased liver mass : body weight ratio in the Alb-cre Tgfbr2flx/flx mice compared to Tgfbr2flx/flx mice. We also observed decreased expression and increased phosphorylation of p130 in the livers from the Alb-cre Tgfbr2flx/flx mice as well as increased expression of cyclin E, which is transcriptionally regulated, in part, by p130:E2F4. Consistent with these results, in a hepatocyte cell line derived from the Tgfbr2flx/flx mice, we found that TGF-β increases the nuclear localization of E2F4, and presumably the transcriptional repression of the p130:E2F4 complex. Thus, we have demonstrated that TGF-β signaling in vivo regulates the mitogenic response in the regenerating liver, affecting the liver mass : body weight ratio after partial hepatectomy, and that these mitogenic responses are accompanied by alterations in p130 expression and phosphorylation, implicating p130 as one of the proteins regulated in vivo by TGF-β during liver regeneration.

Mtgr1 Is a Transcriptional Corepressor That Is Required for Maintenance of the Secretory Cell Lineage in the Small Intestine

ABSTRACT Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.

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.

Short-chain fatty acids inhibit intestinal trefoil factor gene expression in colon cancer cells

Intestinal trefoil factor (ITF) gene expression was detected in five colon cancer cell lines. ITF was synthesized by mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In LIM 1215 cells, the expression of ITF was potently and dose-dependently inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate was ablated by actinomycin D and preceded its effects on differentiation of LIM 1215 cells as indicated by induction of alkaline phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the cyclin D1 gene. Mobility shift assays show specific binding of this response element to nuclear protein extracts of LIM 1215 cells. We conclude that butyrate inhibits ITF expression in colon cancer cells and that this effect may be mediated transcriptionally and independently of its effects on differentiation.Intestinal trefoil factor (ITF) gene expression was detected in five colon cancer cell lines. ITF was synthesized by mucous cells of LIM 1215 and LIM 1863 lines, from which it is secreted constitutively. The ITF mRNA transcript was estimated to be 0.6 kb. In LIM 1215 cells, the expression of ITF was potently and dose-dependently inhibited by short-chain fatty acids (butyrate > propionate > acetate) within 8 h of application. The inhibitory effect of butyrate was ablated by actinomycin D and preceded its effects on differentiation of LIM 1215 cells as indicated by induction of alkaline phosphatase activity and counting of periodic acid-Schiff-positive cells. The human ITF promoter contained an 11-residue consensus sequence with high homology to the butyrate response element of the cyclin D1 gene. Mobility shift assays show specific binding of this response element to nuclear protein extracts of LIM 1215 cells. We conclude that butyrate inhibits ITF expression in colon cancer cells and that this effect may be mediated transcriptionally and independently of its effects on differentiation.

Unlike Esophageal Squamous Cells, Barrett's Epithelial Cells Resist Apoptosis by Activating the Nuclear Factor-κB Pathway

Apoptosis is an important mechanism for maintaining tissue homeostasis and for preventing the proliferation of cells with mutations that could result in malignancy. Barretts epithelium has been reported to be more resistant to apoptosis than normal esophageal squamous epithelium. We have explored the contribution of the nuclear factor-kappaB (NF-kappaB) pathway to apoptotic resistance in non-neoplastic, telomerase-immortalized esophageal squamous (NES) and Barretts (BAR-T) epithelial cell lines. We exposed these cells to UV-B irradiation in doses known to cause DNA damage and to induce apoptosis in normal cells, and studied apoptosis as well as the expression of phospho-H2AX, NF-kappaB, Bcl-2, XIAP, cIAP-1, and survivin proteins. We also used Bay 11-7085 and siRNAs to NF-kappaB and Bcl-2 to assess the effects of NF-kappaB and Bcl2 inhibition on apoptosis. UV-B irradiation at low doses (50 and 100 J/m(2)) caused DNA damage in both NES and BAR-T cells but significantly increased apoptosis only in NES cells. UV-B irradiation caused a decrease in the levels of NF-kappaB, Bcl-2, cIAP-1, XIAP, and survivin in NES cells but increased the levels of those proteins in BAR-T cells. The resistance of BAR-T cells to apoptosis induced by low-dose UV-B irradiation was abolished by Bay 11-7085 and by siRNA for NF-kappaB and was decreased significantly by siRNA for Bcl-2. We conclude that the ability of Barretts epithelial cells to activate the NF-kappaB pathway when they have sustained DNA damage allows them to resist apoptosis. This capacity to avoid apoptosis despite genotoxic damage may underlie the persistence and malignant predisposition of Barretts metaplasia.

Fibrogenesis in pancreatic cancer is a dynamic process regulated by macrophage-stellate cell interaction.

Pancreatic cancer occurs in the setting of a profound fibrotic microenvironment that often dwarfs the actual tumor. Although pancreatic fibrosis has been well studied in chronic pancreatitis, its development in pancreatic cancer is much less well understood. This article describes the dynamic remodeling that occurs from pancreatic precursors (pancreatic intraepithelial neoplasias (PanINs)) to pancreatic ductal adenocarcinoma, highlighting similarities and differences between benign and malignant disease. Although collagen matrix is a commonality throughout this process, early stage PanINs are virtually free of periostin while late stage PanIN and pancreatic cancer are surrounded by an increasing abundance of this extracellular matrix protein. Myofibroblasts also become increasingly abundant during progression from PanIN to cancer. From the earliest stages of fibrogenesis, macrophages are associated with this ongoing process. In vitro co-culture indicates there is cross-regulation between macrophages and pancreatic stellate cells (PaSCs), precursors to at least some of the fibrotic cell populations. When quiescent PaSCs were co-cultured with macrophage cell lines, the stellate cells became activated and the macrophages increased cytokine production. In summary, fibrosis in pancreatic cancer involves a complex interplay of cells and matrices that regulate not only the tumor epithelium but the composition of the microenvironment itself.