Generation and characterization of an inducible transgenic model for studying mouse esophageal biology

ABSTRACT: BACKGROUND: To facilitate the in vivo study of esophageal (stem) cell biology in homeostasis and cancer, novel mouse models are necessary to elicit expression of candidate genes in a tissue-specific and inducible fashion. To this aim, we developed and studied a mouse model to allow labeling of esophageal cells with the histone 2B-GFP (H2B-GFP) fusion protein. RESULTS: First, we generated a transgenic mouse model expressing the reverse tetracycline transactivator rtTA2-M2 under control of the promoter (ED-L2) of the Epstein-Barr virus (EBV) gene encoding the latent membrane protein-1 (LMP-1). The newly generated ED-L2-rtTA2-M2 (ED-L2-rtTA) mice were then bred with the previously developed tetOHIST1H2BJ/GFP (tetO-H2B-GFP) model to assess inducibility and tissue-specificity. Expression of the H2B-GFP fusion protein was observed upon doxycycline induction but was restricted to the terminally differentiated cells above the basal cell layer. To achieve expression in the basal compartment of the esophagus, we subsequently employed a different transgenic model expressing the reverse transactivator rtTA2S-M2 under the control of the ubiquitous, methylation-free CpG island of the human hnRNPA2B1-CBX3 gene (hnRNPrtTA). Upon doxycycline administration to the compound hnRNP-rtTA/tetO-H2B-GFP mice, near-complete labeling of all esophageal cells was achieved. Pulse-chase experiments confirmed that complete turnover of the esophageal epithelium in the adult mouse is achieved within 7-10 days. CONCLUSIONS: We show that the esophagus-specific promoter ED-L2 is expressed only in the differentiated cells above the basal layer. Moreover, we confirmed that esophageal turn-over in the adult mouse does not exceed 7-10 days.     

Novel function of keratins 5 and 14 in proliferation and differentiation of stratified epithelial cells

Keratins are cytoplasmic intermediate filament proteins preferentially expressed by epithelial tissues in a site-specific and differentiation-dependent manner. The complex network of keratin filaments in stratified epithelia is tightly regulated during squamous cell differentiation. Keratin 14 (K14) is expressed in mitotically active basal layer cells, along with its partner keratin 5 (K5), and their expression is down-regulated as cells differentiate. Apart from the cytoprotective functions of K14, very little is known about K14 regulatory functions, since the K14 knockout mice show postnatal lethality. In this study, K14 expression was inhibited using RNA interference in cell lines derived from stratified epithelia to study the K14 functions in epithelial homeostasis. The K14 knockdown clones demonstrated substantial decreases in the levels of the K14 partner K5. These cells showed reduction in cell proliferation and delay in cell cycle progression, along with decreased phosphorylated Akt levels. K14 knockdown cells also exhibited enhanced levels of activated Notch1, involucrin, and K1. In addition, K14 knockdown AW13516 cells showed significant reduction in tumorigenicity. Our results suggest that K5 and K14 may have a role in maintenance of cell proliferation potential in the basal layer of stratified epithelia, modulating phosphatidylinositol 3-kinase/Akt-mediated cell proliferation and/or Notch1-dependent cell differentiation.     

Krüppel-like factor 5 protects against murine colitis and activates JAK-STAT signaling in vivo

Inflammatory bowel disease (IBD), which is characterized by chronic or recurring inflammation of the gastrointestinal tract, affects 1.4 million persons in the United States alone. KLF5, a Krüppel-like factor (KLF) family member, is expressed within the epithelia of the gastrointestinal tract and has been implicated in rapid cell proliferation, migration, and remodeling in a number of tissues. Given these functions, we hypothesized that constitutive Klf5 expression would protect against the development of colitis in vivo. To examine the role of KLF5 in vivo, we used the Villin promoter to target Klf5 to the entire horizontal axis of the small intestine and colon. Villin-Klf5 transgenic mice were born at normal Mendelian ratios and appeared grossly normal to at least 1 year of age. Surprisingly, there were no significant changes in cell proliferation or in the differentiation of any of the intestinal lineages within the duodenum, jejunum, ileum, and colon of Villin-Klf5 mice, compared to littermate controls. However, when Villin-Klf5 mice were treated with dextran sodium sulfate (DSS) to induce colitis, they developed less colonic injury and significantly reduced disease activity scores than littermate controls. The mechanism for this decreased injury may come via JAK-STAT signaling, the activation of which was increased in colonic mucosa of DSS treated Villin-Klf5 mice compared to controls. Thus, KLF5 and its downstream mediators may provide therapeutic targets and disease markers for IBD or other diseases characterized by injury and disruption of intestinal epithelia.     

Impaired NF-kappa B activation and increased production of tumor necrosis factor alpha in transgenic mice expressing keratin K10 in the basal layer of the epidermis

Both the diversity and the precisely regulated tissue- and differentiation-specific expression patterns of keratins suggest that these proteins have specific functions in epithelia besides their well known maintenance of cell integrity. In the search for these specific functions, our previous results have demonstrated that the expression of K10, a keratin expressed in postmitotic suprabasal cells of the epidermis, prevents cell proliferation through the inhibition of Akt kinase activity. Given the roles of Akt in NF-kappa B signaling and the importance of these processes in the epidermis, a study was made into the possible alterations of the NF-kappa B pathway in transgenic mice expressing K10 in the proliferative basal layer. It was found that the inhibition of Akt, mediated by K10 expression, leads to impaired NF-kappa B activity. This appears to occur through the decreased expression of IKK beta and IKK gamma. Remarkably, increased production of tumor necrosis factor alpha and concomitant JNK activation was observed in the epidermis of these transgenic mice. These results confirm that keratin K10 functions in vivo include the control of many aspects of epithelial physiology, which affect the cells not only in a cell autonomous manner but also influence tissue homeostasis.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Abstract. Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: (1) during the differentiation of keratinocytes, (2) during pregnancy. being evident principally in the endometrium but detectable elsewhere as well, and (3) during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.     

Induction of keratinocyte type-I transglutaminase in epithelial cells of the rat

Using immunogold-silver techniques, we have demonstrated that, in rats, type-I (keratinocyte) transglutaminase is expressed primarily in stratified squamous epithelia of the integument, the upper digestive tract, and the lower female genital tract. In these epithelia, the enzyme was found to be present predominantly in the granular layer, but was evident at low levels even in the basal layer, especially in the genital tract. No immunoreactivity was detected in glandular, columnar, or transitional epithelia or in soft tissues. However, considerable enzyme antigenicity was observed in the endometrium and in major ducts of the pancreas and mammary glands of near-term pregnant and early postpartum females. In cultures, substantial immunoreactivity was readily identifiable not only in epidermal, vaginal, and esophageal epithelial cells (immunopositive in vivo), but also in urinary bladder, seminal vesicle, and tracheal epithelial cells (immunonegative in vivo). Primary epithelial outgrowths from bladder and seminal vesicle tissue explants were immunopositive, demonstrating rapid adaptation to the culture environment. These results reveal three distinct levels of regulation of transglutaminase expression in various cell types: during the differentiation of keratinocytes, during pregnancy, being evident principally in the endometrium but detectable elsewhere as well, and during the cultivation of certain epithelia which do not normally express the enzyme in vivo. We conclude that type-I transglutaminase may be a valuable marker for elucidating the regulation of normal epithelial differentiation and squamous metaplasia.     

Selective expansion of the beta-cell compartment in the pancreas of keratinocyte growth factor transgenic mice

Epithelial-mesenchymal interactions are essential for growth, differentiation, and regeneration of exocrine and endocrine cells in the pancreas. The keratinocyte growth factor (KGF) is derived from mesenchyme and has been shown to promote epithelial cell differentiation and proliferation in a paracrine fashion. Here, we have examined the effect of ectopic expression of KGF on pancreatic differentiation and proliferation in transgenic mice by using the proximal elastase promoter. KGF transgenic mice were generated following standard procedures and analyzed by histology, morphometry, immunohistochemistry, Western blot analysis, and glucose tolerance testing. In KGF transgenic mice, the number of islets, the average size of islets, and the relation of endocrine to exocrine tissue are increased compared with littermate controls. An expansion of the beta-cell population is responsible for the increase in the endocrine compartment. Ectopic expression of KGF results in proliferation of beta-cells and pancreatic duct cells most likely through activation of the protein kinase B (PKB)/Akt signaling pathway. Glucose tolerance and insulin secretion are impaired in transgenic animals. These results provide evidence that ectopic expression of KGF in acinar cells promotes the expansion of the beta-cell lineage in vivo through activation of the PKB/Akt pathway. Furthermore, the observed phenotype demonstrates that an increase in the beta-cell compartment does not necessarily result in an improved glucose tolerance in vivo.     

Activated Ras induces lens epithelial cell hyperplasia but not premature differentiation

Growth factor signaling is implicated in the regulation of lens cell proliferation and differentiation during development. Activation of growth factor receptor tyrosine kinases is known to activate Ras proteins, small GTP-binding proteins that function as part of the signal transduction machinery. In the present study, we examined which classical Ras genes are expressed in lens cells during normal development and whether expression of an activated version of Ras is sufficient to induce either lens cell proliferation or fiber cell differentiation in transgenic mice. In situ hybridization showed H-Ras, K-Ras and N-Ras are ubiquitously expressed in all cells of the embryonic (E13.5) eye, with N-Ras showing the highest level of expression. The expression level of N-Ras decreases during later stages of embryonic development, and is nearly undetected in postnatal day 21 lenses. To generate transgenic mice, a constitutively active H-Ras mutant was linked to a chimeric regulatory element containing the mouse alphaA-crystallin promoter fused to the chick delta1-crystallin lens enhancer element. In the lenses of the transgenic mice, the transgene was expressed in both lens epithelial and fiber cells. Expression of activated Ras was sufficient to stimulate lens cell proliferation but not differentiation, implying that alternative or additional signal transduction pathways are required to induce fiber cell differentiation.