The PDX1 Homeodomain Transcription Factor Negatively Regulates the Pancreatic Ductal Cell-specific Keratin 19 Promoter

Abstract

Keratin 19 is a member of the cytokeratin family that is critical for maintenance of cellular architecture and organization, especially of epithelia. The pancreas has three distinct cell types, ductal, acinar, and islet, each with different functions. Embryologically, the pancreatic and duodenal homeobox 1 (PDX1) homeodomain protein is critical for the initiation of all pancreatic lineages; however, the later differentiation of the endocrine pancreas is uniquely dependent upon high PDX1 expression, whereas PDX1 is down-regulated in the ductal and acinar cell lineages. We find that this down-regulation may be required for normal ductal expression of cytokeratin K19. The K19 promoter-reporter gene assay demonstrates that ectopic PDX1 inhibits K19 reporter gene activity in primary pancreatic ductal cells. This is reinforced by our findings that retrovirally mediated stable transduction of PDX1 in primary pancreatic ductal cells suppresses K19 expression, and short interfering RNA to PDX1 in Min6 insulinoma cells results in the induction of normally undetectable K19. Complementary functional and biochemical approaches led to the unexpected finding that a multimeric complex of PDX1 and two members of the TALE homeodomain factor family, MEIS1a and PBX1b, regulates K19 gene transcription through a specific cis-regulatory element (؊341 to ؊325) upstream of the K19 transcription start site. These data suggest a unifying mechanism whereby PDX1, myeloid ecotropic viral insertion site (MEIS), and pre-B-cell leukemia transcription factor 1 (PBX) may regulate ductal and acinar lineage specification during pancreatic development. Specifically, concomitant PDX1 suppression and MEIS isoform expression result in proper ductal and acinar lineage specification. Furthermore, PDX1 may inhibit the ductal differentiation program in the pancreatic endocrine compartment, particularly beta cells.Cytokeratins are members of the intermediate filament family that are critical to the maintenance of cell and tissue integrity (1). In addition, they influence membrane and subcellular localization of proteins. The cytokeratin family consists of at least 20 members that are categorized as acidic type I, comprising keratins 9 -20, or basic type II, comprising keratins 1-8. Typically, cytokeratins form heterodimers between one type I member and one type II member. Keratin 19 (K19) 2 is expressed in epithelia and substitutes for keratin 18 in heterodimerization with keratin 8. Among the pancreatic cell types, K19 is specifically expressed in pancreatic ducts in vivo and in primary pancreatic ductal cells in vitro that our laboratory has successfully isolated and characterized (2). We have previously demonstrated that K19 expression is modulated by the KLF4 and Sp1 zinc-finger transcription factors, contributing to its tissue specificity in the pancreas (3). This activity is mediated by a short cis-regulatory region containing an overlapping binding site for KLF4 and Sp1 within the K19 promoter. KLF4 has a higher binding affinity and is the predominan...

A CK19CreERT knockin mouse line allows for conditional DNA recombination in epithelial cells in multiple endodermal organs

“…K19 encodes an intermediate filament protein (Moll et al, 1982) that is expressed in multiple cell types from the epiblast stage and is maintained in multiple epithelial cell types of later embryonic and postnatal stages (Bosch et al, 1988;Lane et al, 1983;Moll et al, 1982;Quinlan et al, 1985). For example, K19 is highly expressed in the pancreatic ducts of the adult pancreas (Deramaudt et al, 2006), but is absent or weak in acini and islets (Brembeck et al, 2001 We derived a K19 CreERT allele by replacing K19 ATG with a CreER T -cDNA followed by a SV40 polyadenylation signal (see Fig. 1).…”

A feat of metabolic proportions: Pdx1 orchestrates islet development and function in the maintenance of glucose homeostasis

“…Importantly in this regard, Pdx1 expression is often observed in proliferating duct cells [77,94,95], and such cells have been suggested to serve as substrate for β cell neogenesis, particularly in cases of adaptive hyperplasia or pancreas regeneration [92,[96][97][98][99]. A recent study suggests that Pdx1, in complex with TALE transcription factors, negatively regulates the duct-specific keratin 19 gene in mature duct cells [100]. This finding raises the possibility that enhanced levels of Pdx1 protein in duct cells may promote β cell transdifferentiation by direct repression of the duct cell phenotype (through suppression of keratin 19 and related genes) and simultaneous activation of β cell-specific genes [101][102][103].…”

“…Although the majority of these cofactor interactions with Pdx1 are believed to enhance the activation function of Pdx1, there is the distinct possibility that certain cofactor interactions may allow Pdx1 to aid in the repression of some genes. In this regard, the well-defined repression of genes such as glucagon [63,64,70,134] and K19 [100] by Pdx1 may reflect a repression complex formed in association with corepressor molecules.…”

Three-amino-acid-loop-extension homeodomain factor Meis3 regulates cell survival via PDK1

“…Similarly, a Pbx interaction-defective Pdx1 transgene is unable to rescue normal glucose homeostasis in Pdx1 null mice (12). Although Meis/Pbx/Pdx1 complexes regulate gene expression in cultured ductal and acinar cells (2,4), no transcriptional targets of Pbx or Meis have been identified in the β-cell, and whether Meis family members are involved in regulating β-cell development and/or function is still unknown.…”

“…In general, TALE homeodomain factors act via the formation of heterodimeric and -trimeric DNA-binding complexes with one another and/or Hox proteins to activate specific gene expression (1)(2)(3)(4)(5). The TALE homeodomain superclass is composed of Pbx (Pbx1, Pbx2, Pbx3, and Pbx4) and Meis (Prep1, Prep2, Meis1, Meis2, and Meis3) family members in mammals (6).…”