Resident cell populations of the skin contribute to the inflammatory response by producing an array of chemokines, which attract leukocytes from the circulation. TNF-alpha is a major inducer of proinflammatory mediators in <em>keratinocytes</em>. We have recently observed that epidermal <em>growth</em> <em>factor</em> receptor (EGFR) signaling affects TNF-alpha-driven chemokine expression in epidermal <em>keratinocytes</em>, and its functional impairment increases the levels of crucial chemoattractants such as CCL<em>2</em>/MCP-1, CCL5/RANTES, and CXCL10/IFN-gamma-inducible protein-10. In this study, we report evidence that EGFR-dependent ERK1/<em>2</em> activity is implicated in this mechanism. Abrogation of ERK1/<em>2</em> activity with specific inhibitors increased chemokine expression in <em>keratinocytes</em> by enhancing mRNA stabilization. In mouse models, inflammatory response to irritants and T cell-mediated contact hypersensitivity were both aggravated when elicited in a skin area previously treated with an EGFR or a MAPK kinase 1/<em>2</em> inhibitor. In contrast, impairment of p38alpha beta MAPK phosphorylation markedly attenuated these responses. Our data indicate that EGFR-dependent ERK1/<em>2</em> activity in <em>keratinocytes</em> takes part to a homeostatic mechanism regulating inflammatory responses, and emphasize the distinct role of MAPKs as potential targets for manipulating inflammation in the skin.

BACKGROUND

Glucagon-like peptide-<em>2</em> is thought to act as a <em>growth</em> <em>factor</em> for the gut, but the localization of the GLP-<em>2</em> receptor and mechanism of action on epithelial <em>growth</em> is unclear.

RESULTS

We found glucagon-like peptide-<em>2</em> (GLP-<em>2</em>) receptors mainly on subepithelial myofibroblasts in rat, mouse, marmoset and human small and large intestine by immunohistochemistry and in situ hybridisation. By double labelling we found that these GLP-<em>2</em> receptor immunoreactive cells also produce smooth muscle actin and keratinocyte <em>growth</em> <em>factor</em> (KGF). By subcutaneous infusion of either GLP-<em>2</em> alone, GLP-<em>2</em> plus KGF antibody, KGF antibody alone or saline in mice, we found that KGF antibody abolished the <em>growth</em> promoting effect of GLP-<em>2</em> in the large intestine, but not in the small intestine.

CONCLUSIONS

Our findings suggest that GLP-<em>2</em> in the gut acts by activating receptors on the subepithelial myofibroblasts, causing the release of <em>growth</em> <em>factor</em>s, which in turn stimulate intestinal <em>growth</em>.

Epidermal <em>keratinocytes</em> in psoriatic lesions are characterized by activated Stat3, and increased levels of cytokines and <em>growth</em> <em>factors</em> that promote Stat3 activation have been found within psoriatic lesions. K5.Stat3C transgenic mice, in which <em>keratinocytes</em> express a constitutively active Stat3, develop psoriasis-like skin lesions. In this study, we examined whether STA-<em>2</em>1, a small Stat3 inhibitor, could be useful in ameliorating the skin lesions not only in the model mouse but also in human psoriasis. Treatment with STA-<em>2</em>1 markedly inhibited the cytokine-dependent nuclear translocation of Stat3 in normal human <em>keratinocytes</em> in vitro. <em>Keratinocyte</em> proliferation was inhibited by STA-<em>2</em>1 in a dose-dependent manner through downregulation of c-Myc and cyclin D1, whereas involucrin, transglutaminase 1, and keratin 10 levels were upregulated. Topical application of STA-<em>2</em>1 abolished the generation of skin lesions in K5.Stat3C mice. Finally, we treated psoriasis patients with STA-<em>2</em>1-containing ointment in a nonrandomized study. Psoriatic lesions in six of the eight patients showed improvement after topical STA-<em>2</em>1 treatment for <em>2</em> weeks. Therefore, we conclude that targeting Stat3 may lead to a therapy for psoriasis.

OBJECTIVE

Conjugated linoleic acid (CLA) and n-3 polyunsaturated fatty acids (PUFA) have been proposed as important pharmaco-nutrients for modulating mucosal immunity and therapeutic responses in patients with inflammatory bowel disease (IBD). We evaluated the ability of CLA and n-3 PUFA alone or in combination to modulate IBD in a pig model of dextran sodium sulfate (DSS)-induced colitis.

METHODS

Sixty-four, 15-day-old pigs were used to evaluate the effect of CLA, n-3 PUFA and a 50:50 mixture of CLA and n-3 PUFA on growth, clinical activity and colonic PPAR-responsive gene expression. Diets were formulated to contain: 1.33% soybean oil (control); 1.33% CLA; 1.33% fish oil; or 1.33% of a 50:50 mixture of CLA and fish oil. Intestinal inflammation was induced by an intragastric challenge with DSS on day 42 of dietary supplementation. The colonic expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma), PPAR gamma- and delta-responsive genes, keratinocyte growth factor (KGF) and tumor necrosis factor (TNF-alpha) were assayed by quantitative RT-PCR.

RESULTS

The onset of IBD was delayed, colitis less severe and growth suppression attenuated in pigs fed CLA, which correlated with induction of colonic PPAR gamma and its responsive gene PPAR gamma-coactivator-1alpha (PGC1-alpha) and downregulation of TNF-alpha. However, dietary supplementation with n-3 PUFA alone or in combination with CLA resulted in an early onset of disease (i.e., day 2) and faster recovery on days 6 and 7, which correlated with a marked induction of the PPAR delta-responsive gene uncoupling protein 3 (UCP3). CLA and n-3 PUFA acted synergistically to upregulate colonic KGF expression in DSS-challenged pigs but n-3 PUFA blocked CLA-induced PPAR gamma activation.

CONCLUSIONS

Dietary CLA-supplementation upregulated colonic PPAR gamma expression and contributed to delaying the onset of experimental IBD, whereas n-3 PUFA failed to protect from IBD, although it accelerated colonic regeneration and clinical remission by activating PPAR delta.

The Wnt gene family encodes a set of highly conserved secreted signaling proteins that have major roles in embryogenesis and tissue homeostasis. Yet the expression of this family of important mediators in psoriasis, a disease characterized by marked changes in <em>keratinocyte</em> <em>growth</em> and differentiation, is incompletely understood. We subjected 58 paired biopsies from lesional and uninvolved psoriatic skin and 64 biopsies from normal skin to global gene expression profiling. WNT5A transcripts were upregulated fivefold in lesional skin, accompanied by increased Wnt-5a protein levels. Notably, WNT5A mRNA was markedly induced by IL-1alpha, tumor necrosis <em>factor</em>-alpha, IFN-gamma, and transforming <em>growth</em> <em>factor</em>-alpha in cultured <em>keratinocytes</em>. Frizzled <em>2</em> (FZD<em>2</em>) and FZD5, which encode receptors for Wnt5A, were also increased in lesional psoriatic skin. In contrast, expression of WIF1 mRNA, encoding a secreted antagonist of the Wnt proteins, was downregulated >10-fold in lesional skin, along with decreased WNT inhibitory <em>factor</em> (WIF)-1 immunostaining. Interestingly, pathway analysis along with reduced AXIN<em>2</em> expression and lack of nuclear translocation of beta-catenin indicated a suppression of canonical Wnt signaling in lesional skin. The results of our study suggest a shift away from canonical Wnt signaling toward noncanonical pathways driven by interactions between Wnt-5a and its cognate receptors in psoriasis, accompanied by impaired homeostatic inhibition of Wnt signaling by WIF-1 and dickkopf.

Glucagon-like peptide-<em>2</em> (GLP-<em>2</em>) is a pleiotropic hormone that affects multiple facets of intestinal physiology, including <em>growth</em>, barrier function, digestion, absorption, motility, and blood flow. The mechanisms through which GLP-<em>2</em> produces these actions are complex, involving unique signaling mechanisms and multiple indirect mediators. As clinical trials have begun for the use of GLP-<em>2</em> in a variety of intestinal disorders, the elucidation of such mechanisms is vital. The GLP-<em>2</em> receptor (GLP-<em>2</em>R) is a G protein-coupled receptor, signaling through multiple G proteins to affect the cAMP and mitogen-activated protein kinase pathways, leading to both proliferative and antiapoptotic cellular responses. The GLP-<em>2</em>R also demonstrates unique mechanisms for receptor trafficking. Expression of the GLP-<em>2</em>R in discrete sets of intestinal cells, including endocrine cells, subepithelial myofibroblasts, and enteric neurons, has led to the hypothesis that GLP-<em>2</em> acts indirectly through multiple mediators to produce its biological effects. Indeed, several studies have now provided important mechanistic data illustrating several of the indirect pathways of GLP-<em>2</em> action. Thus, insulin-like <em>growth</em> <em>factor</em> I has been demonstrated to be required for GLP-<em>2</em>-induced crypt cell proliferation, likely involving activation of beta-catenin signaling. Furthermore, vasoactive intestinal polypeptide modulates the actions of GLP-<em>2</em> in models of intestinal inflammation, while <em>keratinocyte</em> <em>growth</em> <em>factor</em> is required for GLP-<em>2</em>-induced colonic mucosal <em>growth</em> and mucin expression. Finally, enteric neural GLP-<em>2</em>R signaling affects intestinal blood flow through a nitric oxide-dependent mechanism. Determining how GLP-<em>2</em> produces its full range of biological effects, which mediators are involved, and how these mediators interact is a continuing area of active research.

T cells exercise their full impact on target cells through a combination of secreted cytokines. The recently described T helper cell subset Th<em>2</em><em>2</em> is characterized by a combinatorial secretion of IL-<em>2</em><em>2</em> and TNF-α. Here, we demonstrate that IL-<em>2</em><em>2</em> increases the TNF-α-dependent induction and secretion of several immune-modulatory molecules such as initial complement <em>factors</em> C1r and C1s, antimicrobial peptides S100A7 and HBD-<em>2</em> (human β defensin <em>2</em>), and antimicrobial chemokines CXCL-9/-10/-11 in primary human <em>keratinocytes</em>. The synergism of IL-<em>2</em><em>2</em> and TNF-α is transmitted intracellularly by MAP kinases and downstream by transcription <em>factors</em> of the AP-1 family. The induction of innate immunity is relevant in an in vitro infection model, where <em>keratinocytes</em> stimulated with Th<em>2</em><em>2</em> supernatants or recombinant IL-<em>2</em><em>2</em> plus TNF-α effectively inhibit the <em>growth</em> of Candida albicans and maintain survival of epithelia. Accordingly, the combinatorial stimulation of <em>keratinocytes</em> with IL-<em>2</em><em>2</em> and TNF-α most efficiently conserves the integrity of the epidermal barrier in a three-dimensional skin infection model as compared with IFN-γ, IL-17, IL-<em>2</em><em>2</em> or TNF-α alone. In summary, we demonstrate that IL-<em>2</em><em>2</em> and TNF-α represent a potent, synergistic cytokine combination for cutaneous immunity.

Haem oxygenase (HO) is the rate-limiting enzyme in the degradation of haem. In addition to its obvious role in iron metabolism, a series of findings indicate an important role for HO in cellular protection against oxidative stress. This effect might be of particular importance during wound healing and also in inflammatory disease. Therefore we determined the expression of the two HO isoenzymes, HO-1 and HO-<em>2</em>, during the healing process of full-thickness excisional wounds in mice. We show a remarkable induction of HO-1 mRNA and protein expression within three days after skin injury. After completion of wound healing, HO-1 expression declined to basal levels. By contrast, expression of HO-<em>2</em> was not significantly modulated by skin injury. In situ hybridization and immunohistochemistry revealed high HO-1 expression in inflammatory cells of the granulation tissue and in <em>keratinocytes</em> of the hyperproliferative epithelium. A strong overexpression of HO-1 was also observed in the skin of patients suffering from the inflammatory skin disease psoriasis. In addition, HO-<em>2</em> mRNA levels were increased in the skin of psoriatic patients. Similar to wounded skin, inflammatory cells and <em>keratinocytes</em> of the hyperthickened epidermis were the major producers of HO-1 in psoriatic skin. In vitro studies with cultured <em>keratinocytes</em> revealed a potential role for reactive oxygen species (ROS), but not for <em>growth</em> <em>factors</em> and pro-inflammatory cytokines, as inducers of HO-1 expression in inflamed skin. Our findings suggest a novel role for HO in wound healing and inflammatory skin disease, where it might be involved in haem degradation and in the protection of cells from the toxic effects of ROS.

The expression of genes encoding antioxidant and/or phase <em>2</em> detoxifying enzymes can be enhanced in response to various environmental stresses. The main transcription <em>factor</em> involved in this response is nuclear <em>factor</em> erythroid <em>2</em>-related <em>factor</em> <em>2</em> (Nrf<em>2</em>). Nrf<em>2</em> activity is negatively regulated by the protein Kelch-like-Ech-associated-protein 1 (Keap1). While the roles of Nrf<em>2</em> and phase <em>2</em> genes in chemoprevention of carcinogenesis have been well described; only few studies have dealt with their role in skin cancer. Normal human <em>keratinocytes</em> (NHK) and melanocytes (NHM) were treated by chemical inducers of the Nrf<em>2</em> pathway or by small interfering RNAs (siRNA) used to knock down Keap1 mRNA. The above treatments resulted in significant stimulation of NQO-1 (NADPH-Quinone-Oxidoreductase 1) gene expression. GCL (gamma-Glutamyl-cysteinyl-ligase) gene was also induced but interestingly increased mRNA encoding the catalytic, heavy subunit GCLC was mainly stimulated in NHK, whereas the mRNA encoding the modifier, light subunit GCLM was mostly induced in NHM. HO-1 (Heme Oxygenase 1) gene induction was relatively strong in NHM, but generally absent in NHK, except when the cells were subjected to cytotoxic doses of the above chemicals. Exposure to solar UV (UVB + UVA, 300-400 nm) or to UVA alone (3<em>2</em>0-400 nm) confirmed this trend, but interestingly, at doses where cell <em>growth</em> reduction was comparable, UVA was generally more efficient than solar UV in inducing phase <em>2</em> genes. When siRNAs directed against Nrf<em>2</em> were used, a strong down-regulation of NQO-1 expression was observed in both, NHM and NHK, whereas reduction of HO-1 expression was mainly detected in NHM. To our knowledge, this is the first study comparing phase <em>2</em> gene modulation in NHK and NHM. The results hereby presented should contribute to a better understanding of the molecular mechanisms involved in skin adaptation to environmental stress.

In wound healing epidermal-dermal interactions are known to regulate <em>keratinocyte</em> proliferation and differentiation. To find out how fibroblasts respond to epithelial stimuli, we characterized fibroblasts in monolayer co-culture with <em>keratinocytes</em>. On co-culture numerous extracellular matrix- and smooth muscle cell-associated gene transcripts were up-regulated in fibroblasts, suggesting a differentiation into myofibroblasts. Increased alpha-smooth muscle actin (alpha-SMA) protein expression in co-cultured fibroblasts started at approximately day 4, was serum-independent, but required endogenous transforming <em>growth</em> <em>factor</em> (TGF)-beta. In co-cultures, TGF-beta neutralizing monoclonal antibody strongly reduced alpha-SMA induction. Endogenous TGF-beta production and activation were increased at <em>2</em>4 and 48 hours, requiring, like alpha-SMA induction, close <em>keratinocyte</em>-fibroblast proximity. As myofibroblast differentiation only started after 4 days, we analyzed the presence of endogenous inhibitors at early time points. Blocking <em>keratinocyte</em>-derived interleukin (IL)-1 using IL-1 receptor antagonist, alpha-SMA expression in co-cultures was potentiated. Conversely, adding exogenous IL-1alpha completely suppressed endogenous alpha-SMA induction. In co-cultured fibroblasts strong nuclear <em>factor</em>-kappaB binding activity was observed from <em>2</em> hours, decreasing at <em>2</em> and 4 days, suggesting an early, IL-1-mediated inhibition of TGF-beta signaling in co-cultured fibroblasts. This biphasic differentiation event is regulated by the balance of endogenous TGF-beta and IL-1 activity and is reminiscent of myofibroblast differentiation at early and later stages of wound healing.

We have utilized a broad approach to address whether tyrosine kinases and the <em>growth</em> pathways they regulate might be functionally aberrant in squamous cell carcinomas (SCC) of the upper aerodigestive tract. This strategy involved assaying for evidence of tyrosine kinase action in lysates of cell lines representing SCC. Our findings revealed a spectrum of elevated tyrosine phosphorylation in SCC lines ranging from less than <em>2</em>-fold to more than 10-fold above that of control human epidermal <em>keratinocytes</em>. Thus the ability to regulate <em>growth</em> and other pathways controlled by tyrosine phosphorylation was impaired in all the 19 lines examined. Assessment of the receptor for epidermal <em>growth</em> <em>factor</em> (EGF) revealed that its activity was elevated above normal in 14 of the 19 cell lines examined, suggesting that at least a portion of the increased tyrosine phosphorylation observed could be attributed to excessive EGF receptor activity. Our findings provide functional evidence that <em>growth</em> pathways are aberrantly regulated in cell lines representing SCC of the upper aerodigestive tract.

Distinct from its classic functions in the regulation of calcium and phosphorus metabolism as a systemic hormone, 1alpha,<em>2</em>5-dihydroxyvitamin D(3) [1alpha,<em>2</em>5(OH)(<em>2</em>)D(3)] is involved in the local control and regulation of cellular <em>growth</em> and differentiation in various tissues, including epidermis (<em>keratinocytes</em>) and bone (osteoblasts and osteoclasts). In this review, the impact of 1alpha,<em>2</em>5(OH)(<em>2</em>)D(3) on <em>growth</em> <em>factor</em>/cytokine synthesis and signaling is discussed, particularly as it pertains to bone cells and <em>keratinocytes</em>. 1alpha,<em>2</em>5(OH)(<em>2</em>)D(3) not only regulates <em>growth</em> <em>factor</em>/cytokine synthesis but may also alter <em>growth</em> <em>factor</em> signaling. Recently discovered examples for such interactions are the interactions between the vitamin D receptor and the mothers against decapentaplegic-related proteins that function downstream of TGFbeta receptors. Inhibitory effects of 1alpha,<em>2</em>5(OH)(<em>2</em>)D(3) on <em>keratinocytes</em> through TGFbeta activation and IL-1alpha, IL-6, and IL-8 suppression may provide a rationale for its beneficial effects in the treatment of hyperproliferative skin disorders, whereas stimulatory effects through the epidermal <em>growth</em> <em>factor</em>-related family members and platelet-derived <em>growth</em> <em>factor</em> may be operative in its beneficial effects in skin atrophy and wound healing. Modulation of cytokines and <em>growth</em> <em>factors</em> by 1alpha,<em>2</em>5(OH)(<em>2</em>)D(3) during bone remodeling plays an important role in the coupling of osteoblastic bone formation with osteoclastic resorption to maintain bone mass.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF, FGF-7) is a potent mitogen for epithelial cells. We instilled recombinant human KGF to determine the effects of KGF on alveolar epithelial cells. Left lungs of adult rats were instilled intrabronchially with KGF (5 mg/kg) or normal saline. KGF instillation resulted in epithelial cell hyperplasia, and the alveolar bromodeoxyuridine (BrdU) labeling index peaked at 35% on day <em>2</em> after instillation. The mRNA levels for the surfactant proteins (SPs) SP-A, SP-B, and SP-D were increased in whole lung tissue on days 1 and <em>2</em> after KGF treatment and then returned to control levels on days 3-7. SP-C mRNA levels were increased on days <em>2</em>-5 after KGF instillation. However, all surfactant protein mRNAs were reduced in type II cells isolated from rats instilled with KGF <em>2</em> or 3 days before isolation. These observations were confirmed by in situ hybridization. Instillation of KGF also increased the amount of SP-A and SP-D in lavage fluid. Transcripts for CC10, the 10-kDa Clara cell protein, were decreased. KGF increases the mRNA for the surfactant proteins per lung because of type II cell hyperplasia, but the mRNA per cell is slightly diminished as measured in isolated cells or estimated by in situ hybridization.

Epithelial cells of mucosal tissues provide a barrier against environmental stress, and <em>keratinocytes</em> are key decision makers for immune cell function in the skin. Currently, epithelial signaling networks that instruct barrier immunity remain uncharacterized. Here we have shown that <em>keratinocyte</em>-specific deletion of a disintegrin and metalloproteinase 17 (Adam17) triggers T helper <em>2</em> and/or T helper 17 (Th<em>2</em> and/or Th17) cell-driven atopic dermatitis and myeloproliferative disease. In vivo and in vitro deficiency of ADAM17 dampened Notch signaling, increasing production of the Th<em>2</em> cell-polarizing cytokine TSLP and myeloid <em>growth</em> <em>factor</em> G-CSF. Ligand-independent Notch activation was identified as a regulator of AP-1 transcriptional activity, with Notch antagonizing c-Fos recruitment to the promoters of Tslp and Csf3 (G-CSF). Further, skin inflammation was rescued and myeloproliferation ameliorated by delivery of active Notch to Adam17(-)(/-) epidermis. Our findings uncover an essential role of ADAM17 in the adult epidermis, demonstrating a gatekeeper function of the ADAM17-Notch-c-Fos triad in barrier immunity.

The effects of chitin [(1 ->> 4)-<em>2</em>-acetamido-<em>2</em>-deoxy-beta-D-glucan] and its partially deacetylated derivatives, chitosans, on the proliferation of human dermal fibroblasts and <em>keratinocytes</em> were examined in vitro. Chitosans with relatively high degrees of deacetylation strongly stimulated fibroblast proliferation while samples with lower levels of deacetylation showed less activity. Fraction, CL313A, a shorter chain length, 89% deacetylated chitosan chloride was further evaluated using cultures of fibroblasts derived from a range of human donors. Some fibroblast cultures produced a positive mitogenic response to CL313A treatment with proliferation rates being increased by approximately 50% over the control level at an initial concentration of 50 microg/ml, whilst others showed no stimulation of proliferation or even a slight inhibition (< 10%). The stimulatory effect on fibroblast proliferation required the presence of serum in the culture medium suggesting that the chitosan may be interacting with <em>growth</em> <em>factors</em> present in the serum and potentiating their effect. In contrast to the stimulatory effects on fibroblasts, fraction CL313A inhibited human <em>keratinocyte</em> mitogenesis with up to 40% inhibition of proliferation being observed at 50 microg/ml. In general highly deacetylated chitosans were more active than those with a lower degree of deacetylation. These data demonstrate that highly deacetylated chitosans can modulate human skin cell mitogenesis in vitro. Analysis of their effects on cells in culture may be useful as a screen for their potential activity in vivo as wound healing agents, although in the case of fibroblasts it is important to select appropriate strains of cells for use in the screen.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is important in tissue repair and wound healing and its administration can abrogate chemical- and radiation-induced tissue damage in rodents. We investigated KGF as a therapeutic agent for the prevention of graft-versus-host disease (GVHD)-induced tissue damage, morbidity, and mortality in an established murine allogeneic bone marrow transplantation (BMT) model. B10.BR (H<em>2</em>(k)) recipient mice were lethally irradiated and transplanted with C57BL/6 (H<em>2</em>(b)) bone marrow (BM) with spleen cells (BMS) as a source of GVHD-causing T cells. KGF-treated mice (5 mg/kg/d subcutaneously days -6, -5, and -4 pre-BMT) receiving BMS exhibited better survival than those not receiving KGF (P =.00<em>2</em>7). Cyclophosphamide (Cy), a common component of total body irradiation (TBI)-containing regimens, was administered to other cohorts of mice at a dose of 1<em>2</em>0 mg/kg/d intraperitoneally on days -3 and -<em>2</em> before BMT. KGF-treated mice again exhibited a better survival rate than those not receiving KGF (P =.00086). However, KGF-treated recipients receiving TBI or Cy/TBI BMS were not GVHD-free, as shown by lower body weights compared with BM groups. GVHD target tissues were assessed histologically during a 38-day post-BMT observation period. KGF ameliorated GVHD-induced tissue damage in the liver, skin, and lung (completely in some recipients) and moderately so in the spleen, colon, and ileum, even with Cy conditioning. These studies demonstrate that KGF administration, completed before conditioning, has potential as an anti-GVHD therapeutic agent.

<em>Keratinocytes</em> and other epithelial cells express two receptors for the basement membrane (BM) extracellular matrix component laminin-5 (Ln-5), integrins alpha 3 beta 1 and alpha 6 beta 4. While alpha 3 beta 1 mediates adhesion, spreading, and migration (Kreidberg, J.A. <em>2</em>000. Curr. Opin. Cell Biol. 1<em>2</em>:548--553), alpha 6 beta 4 is involved in BM anchorage via hemidesmosomes (Borradori, L., and A. Sonnenberg. 1999. J. Invest. Dermatol. 11<em>2</em>:411--418). We investigated a possible regulatory interplay between alpha 3 beta 1 and alpha 6 beta 4 in cell motility using HaCaT <em>keratinocytes</em> as a model. We found that alpha 6 beta 4 antibodies inhibit alpha 3 beta 1-mediated migration on Ln-5, but only when migration is haptotactic (i.e., spontaneous or stimulated by alpha 3 beta 1 activation), and not when chemotactic (i.e., triggered by epidermal <em>growth</em> <em>factor</em> receptor). Inhibition of migration by alpha 6 beta 4 depends upon phosphoinositide 3-kinase (PI3-K) since it is abolished by PI3-K blockers and by dominant-negative PI3-K, and constitutively active PI3-K prevents haptotaxis. In HaCaT cells incubated with anti-alpha 6 beta 4 antibodies, activation of PI3-K is mediated by alpha 6 beta 4-associated erbB-<em>2</em>, as indicated by erbB-<em>2</em> autophosphorylation and erbB-<em>2</em>/p85 PI3-K coprecipitation. Furthermore, dominant-negative erbB-<em>2</em> abolishes inhibition of haptotaxis by anti-alpha 6 beta 4 antibodies. These results support a model whereby (a) haptotactic cell migration on Ln-5 is regulated by concerted action of alpha 3beta 1 and alpha 6 beta 4 integrins, (b) alpha 6 beta 4-associated erbB-<em>2</em> and PI3-K negatively affect haptotaxis, and (c) chemotaxis on Ln-5 is not affected by alpha 6 beta 4 antibodies and may require PI3-K activity. This model could be of general relevance to motility of epithelial cells in contact with BM.

OBJECTIVE

Lipoprotein-derived phospholipid oxidation products have been implicated as candidate triggers of the inflammatory process in atherosclerosis. However, in vivo evidence regarding the impact of oxidized phospholipids on the artery wall thus far has been elusive. Therefore, the aim of this study was to investigate if structurally defined oxidized phospholipids induce expression of atherogenic chemokines and monocyte adhesion in intact murine arteries.

RESULTS

To model the accumulation of oxidized phospholipids in the arterial wall, oxidized 1-palmitoyl-<em>2</em>-arachidonoyl-sn-3-glycero-phosphorylcholine (OxPAPC) was topically applied to carotid arteries in mice using pluronic gel. Using quantitative reverse-transcriptase polymerase chain reaction (PCR) and immunohistochemistry, we show that OxPAPC induced a set of atherosclerosis-related genes, including monocyte chemotactic protein 1 (MCP-1) and <em>keratinocyte</em>-derived chemokine (KC), tissue <em>factor</em> (TF), interleukin 6 (IL-6), heme oxygenase 1 (HO-1), and early <em>growth</em> response 1 (EGR-1). OxPAPC-regulated chemokines were also expressed in atherosclerotic lesions of apolipoprotein E-deficient (ApoE-/-) mice. In isolated perfused carotid arteries, OxPAPC triggered rolling and firm adhesion of monocytes in a P-selectin and KC-dependent manner.

CONCLUSIONS

Oxidized phospholipids contribute to vascular inflammation in murine arteries in vivo, initiating atherogenic chemokine expression that leads to monocyte adhesion; therefore, they can be regarded as triggers of the inflammatory process in atherosclerosis.

Myeloablative conditioning results in thymic epithelial cell (TEC) injury, slow T-cell reconstitution, and a high risk of opportunistic infections. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) stimulates TEC proliferation and, when given preconditioning, reduces TEC injury. Thymocytes and TECs express androgen receptors, and exposure to androgen inhibits thymopoiesis. In this study, we have investigated whether TEC stimulation via preconditioning treatment with KGF and leuprolide acetate (Lupron), <em>2</em> clinically approved agents, given only before conditioning would circumvent the profound TEC and associated T-cell deficiency seen in allogeneic bone marrow transplant (BMT) recipients. Only combined treatment with KGF plus leuprolide acetate normalized TEC subset numbers and thymic architecture. Thymopoiesis and thymic output were supranormal, leading to the accelerated peripheral reconstitution of naive CD4 and CD8 T cells with a broad Vbeta repertoire and decreased homeostatic T-cell proliferation. Combined therapy facilitated T:B cooperativity and enabled a B-cell humoral response to a CD4 T cell-dependent neoantigen challenge soon after BMT. In vivo antigen-specific CD8 T-cell responses and clearance of a live pathogen was superior with combined versus individual agent therapy. Thus, KGF combined with androgen blockade represents a novel approach to restore thymic function and facilitates the rapid recovery of peripheral T-cell function after allogeneic BMT.

Ligands of the EGF family regulate autocrine <em>keratinocyte</em> proliferation, and IL-1 family cytokines orchestrate epithelial defense responses. Although members of both families are overexpressed in wound healing and psoriasis, their roles in regulating the innate immune functions of <em>keratinocytes</em> remain incompletely explored. Using sensitive assays, we found significant increases of heparin-binding EGF-like <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-α, and amphiregulin mRNA and protein in lesional psoriasis compared with uninvolved or control skin. In normal human <em>keratinocyte</em> (NHK) monolayers, EGFR ligands were ineffective in inducing DEFB4, S100A7, and CCL<em>2</em>0 mRNAs and human β-defensin (hBD)-<em>2</em> peptide. Combined with IL-1α, however, EGFR ligands provoked <em>2</em>50 × more DEFB4 and CCL<em>2</em>0 and a 9-fold rise in S100A7 mRNA relative to the EGFR ligand alone. This synergy was also reflected in secreted hBD-<em>2</em> protein, both from NHK and reconstituted human epidermis. <em>Keratinocyte</em> differentiation was critical for these responses, as postconfluent NHK yielded mRNA and protein levels an order of magnitude greater than subconfluent cells. Differentiation also influenced signal transduction, with subconfluent cells using NF-κB and postconfluent cells using EGFR, MEK1/<em>2</em>, and p38. We propose that EGFR ligands are important modifiers of IL-1 activity, synergizing with IL-1 to stimulate epidermal production of hBD-<em>2</em>, S100A7, and CCL<em>2</em>0, three of the most upregulated transcripts in psoriatic plaques.

OBJECTIVE

To compare the mRNA expression of growth factor receptors in cultured human trabecular meshwork (HTM) cells with ex vivo HTM tissues and to determine whether HTM cells generate a physiologic response after exposure to exogenous growth factors.

METHODS

The reverse transcription-polymerase chain reaction (RT-PCR) method was used to detect the expression of various growth factor receptor mRNAs using early passaged, cultured HTM cells from donors of several ages. RT-PCR on ex vivo HTM tissues from healthy donors and donors with glaucoma were also used to compare and contrast mRNA expression with cell culture results. After the exogenous administration of growth factors, cell proliferation and extracellular acidification rate studies were used to measure the functional responses of HTM cells to growth factors.

RESULTS

Amplification products of the expected size for 15 growth factor receptors were detected in cultured HTM cells and in ex vivo HTM tissues. The administration of exogenous growth factors showed that (a) hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulinlike growth factor (IGF)-1, tumor necrosis factor (TNF) alpha, platelet-derived growth factor (PDGF)-AA, PDGF-BB, PDGF-AB, and basic fibroblast growth factor (FGF-2) stimulated cell proliferation, whereas FGF-1 (acidic), transforming growth factor (TGF) alpha, interleukin (IL)-1alpha, nerve growth factor (NGF), and FGF-7 (keratinocyte growth factor [KGF]) had no significant influence on cell proliferation; (b) TGF-beta isoforms significantly inhibited EGF-stimulated trabecular meshwork cell proliferation; and (c) FGF-1 (acidic), TGF-alpha, EGF, IL-1alpha, IL-1beta, HGF, TNF-alpha, PDGF-AA, and IGF-1 significantly stimulated extracellular acidification, whereas FGF-2 (basic), FGF-7 (KGF), TGF-beta1-beta3 and NGF had no significant influence on extracellular acidification.

CONCLUSIONS

These studies show that mRNA for numerous growth factor receptors can be detected in cultured HTM cells and in ex vivo HTM tissues. They also show that many of the receptors are functional, because exogenous growth factor administration elicits a physiologic response. In vivo, these receptors may be activated by growth factors present within the aqueous humor (aquecrine/paracrine) or by growth factors synthesized and released locally by trabecular meshwork cells themselves (autocrine). Specific growth factors acting through high-affinity receptors may be involved in maintaining the normal microenvironment of the HTM and also may be involved in the pathogenesis of primary open-angle glaucoma.

Fibroblast growth factors (FGFs) are master regulators of organogenesis and tissue homeostasis. In this study, we used different combinations of FGF receptor (FGFR)-deficient mice to unravel their functions in the skin. Loss of the IIIb splice variants of FGFR1 and FGFR2 in keratinocytes caused progressive loss of skin appendages, cutaneous inflammation, keratinocyte hyperproliferation, and acanthosis. We identified loss of FGF-induced expression of tight junction components with subsequent deficits in epidermal barrier function as the mechanism underlying the progressive inflammatory skin disease. The defective barrier causes activation of keratinocytes and epidermal gammadelta T cells, which produce interleukin-1 family member 8 and S100A8/A9 proteins. These cytokines initiate an inflammatory response and induce a double paracrine loop through production of keratinocyte mitogens by dermal cells. Our results identify essential roles for FGFs in the regulation of the epidermal barrier and in the prevention of cutaneous inflammation, and highlight the importance of stromal-epithelial interactions in skin homeostasis and disease.

BACKGROUND

RNA interference (RNAi) screens have been used to identify novel components of signal-transduction pathways in a variety of organisms. We performed a small interfering (si)RNA screen for novel members of the transforming growth factor (TGF)-β pathway in a human keratinocyte cell line. The TGF-β pathway is integral to mammalian cell proliferation and survival, and aberrant TGF-β responses have been strongly implicated in cancer.

RESULTS

We assayed how strongly single siRNAs targeting each of 6,000 genes affect the nuclear translocation of a green fluorescent protein (GFP)-SMAD2 reporter fusion protein. Surprisingly, we found no novel TGF-β pathway members, but we did find dominant off-target effects. All siRNA hits, whatever their intended direct target, reduced the mRNA levels of two known upstream pathway components, the TGF-β receptors 1 and 2 (TGFBR1 and TGFBR2), via micro (mi)RNA-like off-target effects. The scale of these off-target effects was remarkable, with at least 1% of the sequences in the unbiased siRNA library having measurable off-target effects on one of these two genes. It seems that relatively minor reductions of message levels via off-target effects can have dominant effects on an assay, if the pathway output is very dose-sensitive to levels of particular pathway components. In search of mechanistic details, we identified multiple miRNA-like sequence characteristics that correlated with the off-target effects. Based on these results, we identified miR-20a, miR-34a and miR-373 as miRNAs that inhibit TGFBR2 expression.

CONCLUSIONS

Our findings point to potential improvements for miRNA/siRNA target prediction methods, and suggest that the type II TGF-β receptor is regulated by multiple miRNAs. We also conclude that the risk of obtaining misleading results in siRNA screens using large libraries with single-assay readout is substantial. Control and rescue experiments are essential in the interpretation of such screens, and improvements to the methods to reduce or predict RNAi off-target effects would be beneficial.

Palifermin, a recombinant human <em>keratinocyte</em> <em>growth</em> <em>factor</em>, was tested for potential benefits on acute graft-versus-host disease (GVHD) and hematopoietic recovery in allogeneic hematopoietic stem cell transplantation (HSCT) recipients. This randomized, double-blind, placebo-controlled, dose-escalation study assessed the safety and tolerability of palifermin (n = 69) as compared with placebo (n = 31) in patients conditioned with cyclophosphamide and fractionated total-body irradiation (Cy/TBI) or busulfan and cyclophosphamide (Bu/Cy) and given methotrexate along with a calcineurin inhibitor (cyclosporine A, tacrolimus) for GVHD prophylaxis. All patients received 3 doses before conditioning and either 3 (cohort 1), 6 (cohort <em>2</em>), or 9 (cohort 3) doses after HSCT. Palifermin doses were 40 mug/kg per day (cohort 1 only) or 60 mug/kg per day (all cohorts). Six patients (placebo = <em>2</em>, palifermin = 4) experienced a total of 11 dose-limiting toxicities (most often skin, respiratory, or oral mucositis). The most common adverse events included edema, infection, skin pain, or rash. Times to neutrophil and platelet engraftment were similar. No significant differences in acute GVHD incidence or severity, survival, or day 100 relapse rates were observed between groups. Palifermin was associated with reduced incidence and mean severity of mucositis in patients conditioned with Cy/TBI but not Bu/Cy. We conclude that palifermin was generally safe in allogeneic HSCTs but had no significant effect on engraftment, acute GVHD, or survival in this trial.