Glucagon-like peptide 2 (GLP-2) is a 33-aa proglucagon-derived peptide produced by intestinal enteroendocrine cells. GLP-2 stimulates intestinal growth and upregulates villus height in the small intestine, concomitant with increased crypt cell proliferation and decreased enterocyte apoptosis. Moreover, GLP-2 prevents intestinal hypoplasia resulting from total parenteral nutrition. However, the mechanism underlying these actions has remained unclear. nM). GLP-2 analogs that activated GLP-2R signal transduction in vitro displayed intestinotrophic activity in vivo.These results strongly suggest that GLP-2, like glucagon and GLP-1, exerts its actions through a distinct and specific novel receptor expressed in its principal target tissue, the gastrointestinal tract.Glucagon-like peptides (GLPs) encoded by the proglucagon gene play key roles in glucose homeostasis, gastric emptying, insulin secretion, and appetite regulation (1). Glucagon and GLP-1 exert their effects through distinct G protein-coupled receptors (GPCRs). In contrast, unique receptors for GLP-2, glicentin, and oxyntomodulin have not yet been identified, despite considerable attempts at receptor isolation via classical molecular biology approaches (2). Recent studies have shown that GLP-2 is a potent intestinal growth factor that stimulates crypt cell proliferation and inhibits epithelial apoptosis (3). GLP-2 promotes epithelial proliferation in both small and large intestine; however, the mechanisms utilized by GLP-2 for promotion of intestinal growth remain unclear.To understand the mechanisms underlying GLP-2 action, we have carried out studies directed at the identification and cloning of the putative GLP-2 receptor. We now have isolated rat and human cDNAs encoding GLP-2-responsive GPCRs, which show highest similarity to receptors for glucagon and GLP-1. The GLP-2R is coupled to activation of adenylate cyclase, and the receptor is expressed selectively in rat hypothalamus and the gastrointestinal tract, known targets of GLP-2 action. These findings establish GLP-2 as a novel hormone that, like glucagon and GLP-1, exerts its actions through a distinct receptor expressed in a highly tissuerestricted manner. The GLP-2R should provide an important target for isolation of small molecules mimicking GLP-2 action and for future studies delineating specific mechanisms underlying GLP-2 action in the gut and central nervous system.

Rhamm (receptor for hyaluronan-mediated motility) is an hyaluronan binding protein with limited expression in normal tissues and high expression in advanced cancers. To understand its physiological functions and identify the molecular mechanisms underlying these functions, we created mice with a genetic deletion of Rhamm. We show that Rhamm−/− fibroblasts fail to resurface scratch wounds >3 mm or invade hyaluronan-supplemented collagen gels in culture. We identify a requirement for Rhamm in the localization of CD44 to the cell surface, formation of CD44–ERK1,2 (extracellular-regulated kinase 1,2) complexes, and activation/subcellular targeting of ERK1,2 to the cell nucleus. We also show that cell surface Rhamm, restricted to the extracellular compartment by linking recombinant protein to beads, and expression of mutant active mitogen-activated kinase kinase 1 (Mek1) are sufficient to rescue aberrant signaling through CD44–ERK1,2 complexes in Rh−/− fibroblasts. ERK1,2 activation and fibroblast migration/differentiation is also defective during repair of Rh−/− excisional skin wounds and results in aberrant granulation tissue in vivo. These results identify Rhamm as an essential regulator of CD44–ERK1,2 fibroblast motogenic signaling required for wound repair.

To determine the role of CD4 molecules in the generation and regulation of contact hypersensitivity (CHS), we treated mice lacking the CD4 gene as a result of targeted disruption with dinitrofluorobenzene to induce CHS. The mutant mice lacking CD4 (CD4(-) mice) showed marked hyporesponsiveness in CHS compared with normal syngeneic C57BL/6 mice (38.3 +/-9.0% of normal at 24 h after the challenge assessed by net ear swelling; p < 0.025). CD4(-) mice had a larger CD4-8- double negative T-cell receptor alpha beta+ cell population in the lymph nodes than did normal mice, and the increase of this cell population was observed in CD4(-) mice after sensitization. Draining lymph node cells from sensitized normal mice restored the responsiveness in CD4(-) mice, but those from sensitized CD4(-) mice were less effective in restoring the CHS response in normal mice. Langerhans cell numbers were normal, and function, as assessed by the ability to present soluble hapten, was not impaired in CD4(-) mice. Skin cytokine profiles demonstrated an increase in interferon-gamma, interleukin-2, and interleukin-4 mRNA levels after challenge in normal mice, whereas this response was blunted in CD4(-) mice. CD4(-) mice also showed hyporesponsiveness in inflammatory reaction to irritant chemicals. These results suggest that the CD4 molecule is required for optimal induction of CHS as well as irritant contact dermatitis and may influence the development of CHS by modulating the cytokine profiles in the skin.

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Transforming growth factor-beta (TGF-beta) plays an important role not only in cell growth control but also in inflammation and immunoregulation. There are at least five different isoforms of TGF-beta. TGF-beta 1 has a large variety of biological functions including the modulation of inflammation and the immune system and has most extensively been studied in skin. Since ultraviolet B (UVB) is known to induce skin erythema and immunosuppression, we sought to examine whether UVB would alter the expression and production of TGF-beta 1 in normal human keratinocytes. Using reverse transcription-polymerase chain reaction (RT-PCR), constitutive expression of TGF-beta 1 mRNA was detected in keratinocytes and the level of TGF-beta 1 mRNA was increased 4 and 8 h after 300 J/m2 UVB irradiation. Production of TGF-beta 1 protein in culture supernatants assayed by ELISA was also increased at 24 h after irradiation. Cycloheximide treatment blocked this TGF-beta 1 protein induction indicating de novo protein synthesis of TGF-beta 1 from keratinocytes induced by UVB. These results suggest a possible role for TGF-beta 1 in UVB-induced skin inflammation and immunosuppression.

Keratinocytes are known to produce a wide variety of cytokines which are believed to play a significant role in cutaneous inflammatory and immunologic reactions. Considering the array of proteolytic enzymes present in the skin and the transient nature of cytokines produced from keratinocytes, it is unclear whether cytokines released by keratinocytes cross the basement membrane and contribute to distal inflammatory and immune reactions. To investigate the ability of cytokines released from human keratinocytes to cross basement membrane, we used a two chamber culture model. Keratinocytes were plated in the upper chamber coated with a reconstituted basement membrane matrix (matrigel) on a microporous membrane. To augment cytokine production, we exposed keratinocytes to 300 J/m2 UVB; 24 h later the supernatants were collected, and the levels of cytokine were measured by ELISA. IL-1alpha, IL-6, and TNF-alpha were found to be increased after UVB irradiation in the upper chamber, and significant amounts (70-80%) of each cytokine were detected in the lower chamber. Our results indicate that keratinocyte-derived cytokines are available for interactions below the basement membrane and present circumstantial evidence that the production of those cytokines from keratinocytes contributes to the elevation of circulation after the UVB exposure.

Keratinocyte growth factor (KGF) and its receptor (KGFR) are thought to play important roles in normal keratinocyte growth and differentiation. Since UVB radiation is known to influence keratinocyte growth, we sought to determine whether UVB would alter the expression of KGF and KGFR. Using a reverse-transcription coupled polymerase chain reaction (RT-PCR), the present study examined the expression of KGF and KGFR mRNA in cultured normal human keratinocytes exposed to UVB irradiation. Total cellular RNA was extracted from cultured keratinocytes at various time points after irradiation, reverse transcribed and used for PCR amplification using primers specific for KGF and KGFR. Constitutive expression of KGFR mRNA, but not KGF mRNA, was detected in normal cultured human keratinocytes. After UVB irradiation at 300 J/m2, the KGF mRNA remained undetectable while the KGFR mRNA level was significantly decreased. The down-regulation of KGFR mRNA expression was also confirmed by Northern blot analysis. Immunohistochemical studies demonstrated a decreased positive signal of KGFR in human keratinocytes after UVB irradiation. Our results suggest a possible role for the KGF-KGFR signalling pathway in the skin after exposure to UVB, and that UVB-induced growth inhibition of keratinocytes in hyperproliferative skin disorders may be related to downregulation of KGFR.

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