Onset of keratin 17 expression coincides with the definition of major epithelial lineages during skin development.

The type I keratin 17 (K17) shows a peculiar localization in human epithelial appendages including hair follicles, which undergo a growth cycle throughout adult life. Additionally K17 is induced, along with K6 and K16, early after acute injury to human skin. To gain further insights into its potential function(s), we cloned the mouse K17 gene and investigated its expression during skin development. Synthesis of K17 protein first occurs in a subset of epithelial cells within the single-layered, undifferentiated ectoderm of embryonic day 10.5 mouse fetuses. In the ensuing 48 h, K17-expressing cells give rise to placodes, the precursors of ectoderm-derived appendages (hair, glands, and tooth), and to periderm. During early development, there is a spatial correspondence in the distribution of K17 and that of lymphoid-enhancer factor (lef-1), a DNA-bending protein involved in inductive epithelial-mesenchymal interactions. We demonstrate that ectopic lef-1 expression induces K17 protein in the skin of adult transgenic mice. The pattern of K17 gene expression during development has direct implications for the morphogenesis of skin epithelia, and points to the existence of a molecular relationship between development and wound repair.

Ectopic expression of Hel-N1, an RNA-binding protein, increases glucose transporter (GLUT1) expression in 3T3-L1 adipocytes.

3T3-L1 preadipocytes ectopically expressing the mammalian RNA-binding protein Hel-N1 expressed up to 10-fold more glucose transporter (GLUT1) protein and exhibited elevated rates of basal glucose uptake. Hel-N1 is a member of the ELAV-like family of proteins associated with the induction and maintenance of differentiation in various species. ELAV proteins are known to bind in vitro to short stretches of uridylates in the 3' untranslated regions (3'UTRs) of unstable mRNAs encoding growth-regulatory proteins involved in transcription and signal transduction. GLUT1 mRNA also contains a large 3'UTR with a U-rich region that binds specifically to Hel-N1 in vitro. Analysis of the altered GLUT1 expression at the translational and posttranscriptional levels suggested a mechanism involving both mRNA stabilization and accelerated formation of translation initiation complexes. These findings are consistent with the hypothesis that the Hel-N1 family of proteins modulate gene expression at the level of mRNA in the cytoplasm.

Glucose transporter gene expression: regulation of transcription and mRNA stability.

The facilitated diffusion of D-glucose across the plasma membrane is carried out by a set of stereospecific transport proteins known as the glucose transporters. These integral membrane proteins are members of a gene family where tissue-specific expression of one or more members will determine in part the net rate of glucose entry into the cell. The regulation of glucose transporter gene expression is a critical feature of cellular homeostasis, as defects in specific transporter expression can lead to profound alterations in cellular physiology. In this review, we provide a brief descriptive background on the family of glucose transporters and examine in depth the regulation of the two transporters expressed in adipose tissue, GLUTI, a basal growth-related transporter and GLUT4, the insulin-responsive glucose transporter.

Keratin 17 expression in the hard epithelial context of the hair and nail, and its relevance for the pachyonychia congenita phenotype.

The hard-keratin-containing portion of the murine hair shaft displays a positive immunoreactivity with an antibody against the soft epithelial keratin, K17. The K17-expressing cell population is located in the medulla compartment of the hair. Consistent with this observation, K17-containing cells also occur in the presumptive medulla precursor cells located in the hair follicle matrix. Western blot analysis of hair extracts prepared from a number of mouse strains confirms this observation and suggests that K17 expression in the hair shaft is a general trait in this species. The expression of K17 in human hair extracts is restricted to eyebrow and facial hair samples. These are the major sites for the occurrence of the pili torti (twisted hair) phenotype in the type 2 (Jackson-Lawler) form of pachyonychia congenita, previously shown to arise from inherited K17 mutations. Given that all forms of pachyonychia congenita show an involvement of the nail, we compared the expression of the two other genes mutated in pachyonychia congenita diseases, K6 and K16, with that of K17 in human nail. All three keratins are abundantly expressed within the nail bed epithelium, whereas K17 protein is expressed in the nail matrix, which contains the epithelial cell precursors for the nail plate. Our data suggest a role for K17 in the formation and maintenance of various skin appendages and directly support the concept that pachyonychia congenita is a disease of the nail bed.

Altered radioprotective properties of interleukin I alpha (IL-1) in non-hematologic tumor-bearing animals.

The radioprotective properties of IL-1 were investigated in the respective murine hosts for the Lewis lung (LLca) and EMT-6 tumors. For these studies, doses of total body irradiation were selected for the C57B1/6 (9.5 Gy) and Balb/c (7.5 Gy) mice that resulted in a 60% mortality over a 30-day interval. When a "priming" dose of 2.5 x 10(5) U IL-1 was administered 24 hr prior to the radiation exposure, animal mortality was markedly reduced (60% vs 5-10%). Under identical experimental conditions, however, the presence of either the LLca or the EMT-6 tumors in their respective host strains was found to compromise the level of radioprotection conferred by this priming dose of IL-1. In Balb/c mice bearing the EMT-6 tumor, a priming dose of IL-1 resulted in only a modest level of radioprotection when compared to non-tumor-bearing control animals (median animal survival increased by 11.5 days). In C57B1/6 mice bearing the LLca tumor, IL-1 failed to demonstrate any evidence of radioprotection. Following a sublethal dose of total body irradiation, the appearance of an accelerated repopulation of the stem cell (8d CFUs and CFU-GEMM) and the myeloid progenitor (CFU-M) compartment in the marrow of the IL-1 primed EMT-6, but not the LLca, tumor-bearing animals was consistent with the hypothesis that the mechanism leading to radioprotection in IL-1 primed rodents involves an accelerated recovery of hematopoietic activity. It was also noted that the presence of the EMT-6 tumor was associated with an increase in the "radiosensitivity" of the Balb/c mouse. Collectively, these data suggest that the use of biological modifiers should be examined under altered physiological conditions prior to attempting to translate them into the clinic.

Tumor necrosis factor-alpha regulation of glucose transporter (GLUT1) mRNA turnover. Contribution of the 3'-untranslated region of the GLUT1 message.

In the current study we report on the contribution of the GLUT1 3'-untranslated region (UTR) to the stability of the GLUT1 mRNA. To facilitate these investigations, a hybrid construct was prepared by insertion of the GLUT1 3'-UTR into a normally stable reporter gene coding for preproinsulin. The GLUT1 3'-UTR conferred lability to the otherwise long lived construct and transferred an ability to be stabilized in response to treatment with the cytokine, tumor necrosis factor-alpha (TNF). The destabilizing element has been mapped to a region located between bases 2242 and 2347 of the GLUT1 3'-UTR; this same region also mediates the stabilization response to TNF. In vitro RNA-protein binding assays using protein extracts from control and TNF-treated cells demonstrated that two proteins, one of 37 kDa and the other of 40 kDa, recognized sequence elements within the stability-determining region and were up-regulated in response to TNF treatment. The RNA-binding activity of these proteins coincides with the stabilization of the GLUT1 message, suggesting that they may be involved in regulation of the turnover of this message.

Hel-N1, an RNA-binding protein, is a ligand for an A + U rich region of the GLUT1 3' UTR.

Hel-N1, is an RRM protein which is a mammalian homologue of the Drosophila melanogaster RNA binding protein, ELAV (embryonic lethal abnormal vision). Hel-N1 binds to RNA containing short stretches of uridylates similar to those found in the 3' untranslated regions (3'-UTRs) of oncoprotein and cytokine mRNAs. The GLUT1 glucose transporter has an extensive 3' UTR that is AU-rich reminiscent of the 3'UTR of an oncogene mRNA. An in vitro RNA binding assay using Hel-N1 demonstrated binding to a specific portion of the GLUT1 3'UTR. Analysis of the folding pattern of this region depicted the retention of a stem loop structure, wherein the loop is composed of a stretch of uridylates. To further analyze the potential function of Hel-N1, stable transfectants were made in the 3T3-L1 cell line. The transfectants have been characterized, and the presence of the Hel-N1 DNA and protein verified. Data indicate Hel-N1 is a ligand for GLUT1 and its binding affects the stability and translatability of the GLUT1 message.

Arachidonic acid down-regulates the insulin-dependent glucose transporter gene (GLUT4) in 3T3-L1 adipocytes by inhibiting transcription and enhancing mRNA turnover.

Chronic exposure of fully differentiated 3T3-L1 adipocytes to 50 microM arachidonic acid (AA) resulted in an inhibition (approximately 91%) in cellular GLUT4 mRNA content after a 48-h exposure, without similarly affecting the mRNA content of the ubiquitous glucose transporter, GLUT1. Subsequent investigations revealed that transcription of the GLUT4 gene was reduced by approximately 50% in response to AA treatment and the half-life of GLUT4 mRNA decreased from 8.0 to 4.6 h. By contrast, AA increased the accumulation of GLUT1 mRNA by 65%, by a mechanism that also involved regulation at both transcriptional and mRNA stability levels. Western blot analysis revealed that AA was specifically reducing the insulin-responsive glucose transporter (GLUT4) in both plasma and intracellular membranes. Subsequently, AA was observed to alter the ability of the GLUT4 transporter to respond to insulin and mediate a significant enhancement of glucose uptake. The results presented in this study indicate that AA can partially mimic the effects of both tumor necrosis factor-alpha and insulin which, when chronically supplied to 3T3-L1 adipocytes, also down-regulate GLUT4 gene expression. Therefore, these data may have relevance to the insulin-resistance associated with non-insulin-dependent diabetes mellitus.