Th17 cells are a distinct lineage of effector CD4(+) T cells characterized by their production of interleukin (IL)-17. We demonstrate that Th17 cells also expressed IL-<em>2</em><em>2</em>, an IL-10 family member, at substantially higher amounts than T helper (Th)1 or Th<em>2</em> cells. Similar to IL-17A, IL-<em>2</em><em>2</em> expression was initiated by transforming <em>growth</em> <em>factor</em> beta signaling in the context of IL-6 and other proinflammatory cytokines. The subsequent expansion of IL-<em>2</em><em>2</em>-producing cells was dependent on IL-<em>2</em>3. We further demonstrate that IL-<em>2</em><em>2</em> was coexpressed in vitro and in vivo with both IL-17A and IL-17F. To study a functional relationship among these cytokines, we examined the expression of antimicrobial peptides by primary <em>keratinocytes</em> treated with combinations of IL-<em>2</em><em>2</em>, IL-17A, and IL-17F. IL-<em>2</em><em>2</em> in conjunction with IL-17A or IL-17F synergistically induced the expression of beta-defensin <em>2</em> and S100A9 and additively enhanced the expression of S100A7 and S100A8. Collectively, we have identified IL-<em>2</em><em>2</em> as a new cytokine expressed by Th17 cells that synergizes with IL-17A or IL-17F to regulate genes associated with skin innate immunity.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) inhibits cell proliferation by inducing a G1-phase cell cycle arrest. Normal progression through G1 is promoted by the activity of the cyclin-dependent protein kinases CDK4 and CDK6 (ref. <em>2</em>), which are inhibited by the protein p16INK4. We have isolated a new member of the p16INK4 family, p15INK4B. p15 expression is induced approximately 30-fold in human <em>keratinocytes</em> by treatment with TGF-beta, suggesting that p15 may act as an effector of TGF-beta-mediated cell cycle arrest. The gene encoding p15 is located on chromosome 9 adjacent to the p16 gene at a frequent site of chromosomal abnormality in human tumours (9p<em>2</em>1).

In the last few years, microRNAs (miRNA) have started a revolution in molecular biology and emerged as key players in the carcinogenesis. They have been identified in various tumor types, showing that different sets of miRNAs are usually deregulated in different cancers. To identify the miRNA signature that was specific for oral squamous cell carcinoma (OSCC), we first examined expression profiles of 148 miRNAs in a panel of 18 OSCC cell lines and the immortalized oral <em>keratinocyte</em> line RT7 as a control. Compared with RT7, the expression of 54 miRNAs (36.5%) was frequently down-regulated in OSCC lines (<0.5-fold expression,>>or=66.7% of 18 lines). Among these 54 miRNAs, we further analyzed four of these miRNAs (i.e., miR-34b, miR-137, miR-193a, and miR-<em>2</em>03), located around CpG islands, to identify tumor-suppressive miRNAs silenced through aberrant DNA methylation. The expression of those four genes was restored by treatment with 5-aza-<em>2</em>'-deoxycytidine in OSCC cells lacking their expression. In addition, expression levels of the four miRNAs were inversely correlated with their DNA methylation status in the OSCC lines. In primary tumors of OSCC with paired normal oral mucosa, down-regulation of miRNA expression through tumor-specific hypermethylation was more frequently observed for miR-137 and miR-193a than for miR-34b and miR-<em>2</em>03. Moreover, the ectopic transfection of miR-137 or miR-193a into OSCC lines lacking their expressions significantly reduced cell <em>growth</em>, with down-regulation of the translation of cyclin-dependent kinase 6 or E<em>2</em>F transcription <em>factor</em> 6, respectively. Taken together, our results clearly show that miR-137 and miR-193a are tumor suppressor miRNAs epigenetically silenced during oral carcinogenesis.

Persistent microvascular hyperpermeability to plasma proteins even after the cessation of injury is a characteristic but poorly understood feature of normal wound healing. It results in extravasation of fibrinogen that clots to form fibrin, which serves as a provisional matrix and promotes angiogenesis and scar formation. We present evidence indicating that vascular permeability <em>factor</em> (VPF; also known as vascular endothelial <em>growth</em> <em>factor</em>) may be responsible for the hyperpermeable state, as well as the angiogenesis, that are characteristic of healing wounds. Hyperpermeable blood vessels were identified in healing split-thickness guinea pig and rat punch biopsy skin wounds by their capacity to extravasate circulating macromolecular tracers (colloidal carbon, fluoresceinated dextran). Vascular permeability was maximal at <em>2</em>-3 d, but persisted as late as 7 d after wounding. Leaky vessels were found initially at the wound edges and later in the subepidermal granulation tissue as <em>keratinocytes</em> migrated to cover the denuded wound surface. Angiogenesis was also prominent within this 7-d interval. In situ hybridization revealed that greatly increased amounts of VPF mRNA were expressed by <em>keratinocytes</em>, initially those at the wound edge, and, at later intervals, <em>keratinocytes</em> that migrated to cover the wound surface; occasional mononuclear cells also expressed VPF mRNA. Secreted VPF was detected by immunofluoroassay of medium from cultured human <em>keratinocytes</em>. These data identify <em>keratinocytes</em> as an important source of VPF gene transcript and protein, correlate VPF expression with persistent vascular hyperpermeability and angiogenesis, and suggest that VPF is an important cytokine in wound healing.

Expression cDNA cloning and structural analysis of the human <em>keratinocyte</em> <em>growth</em> <em>factor</em> receptor (KGFR) revealed identity with one of the fibroblast <em>growth</em> <em>factor</em> (FGF) receptors encoded by the bek gene (FGFR-<em>2</em>), except for a divergent stretch of 49 amino acids in their extracellular domains. Binding assays demonstrated that the KGFR was a high-affinity receptor for both KGF and acidic FGF, while FGFR-<em>2</em> showed high affinity for basic and acidic FGF but no detectable binding by KGF. Genomic analysis of the bek gene revealed two alternative exons responsible for the region of divergence between the two receptors. The KGFR transcript was specific to epithelial cells, and it appeared to be differentially regulated with respect to the alternative FGFR-<em>2</em> transcript. Thus, two <em>growth</em> <em>factor</em> receptors with different ligand-binding specificities and expression patterns are encoded by alternative transcripts of the same gene.

We previously found that human mesenchymal stem cells (MSC) or its conditioned medium restored lung protein permeability and reduced alveolar inflammation following Escherichia coli endotoxin-induced acute lung injury (ALI) in an ex vivo perfused human lung in part through the secretion of soluble <em>factors</em> such as <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF). Recently, MSC were found to release microvesicles (MVs) that were biologically active because of the presence of mRNA or miRNA with reparative properties. MVs are circular fragments of membrane released from the endosomal compartment as exosomes or shed from the surface membranes. These studies were designed to determine if MVs released by human bone marrow derived MSCs would be effective in restoring lung protein permeability and reducing inflammation in E. coli endotoxin-induced ALI in C57BL/6 mice. The intratracheal instillation of MVs improved several indices of ALI at 48 hours. Compared to endotoxin-injured mice, MVs reduced extravascular lung water by 43% and reduced total protein levels in the bronchoalveolar lavage (BAL) fluid by 35%, demonstrating a reduction in pulmonary edema and lung protein permeability. MVs also reduced the influx of neutrophils and macrophage inflammatory protein-<em>2</em> levels in the BAL fluid by 73% and 49%, respectively, demonstrating a reduction in inflammation. KGF siRNA-pretreatment of MSC partially eliminated the therapeutic effects of MVs released by MSCs, suggesting that KGF protein expression was important for the underlying mechanism. In summary, human MSC-derived MVs were therapeutically effective following E. coli endotoxin-induced ALI in mice in part through the expression of KGF mRNA in the injured alveolus.

Vascular endothelial <em>growth</em> <em>factor</em> receptor-3 (VEGFR-3) has an essential role in the development of embryonic blood vessels; however, after midgestation its expression becomes restricted mainly to the developing lymphatic vessels. The VEGFR-3 ligand VEGF-C stimulates lymphangiogenesis in transgenic mice and in chick chorioallantoic membrane. As VEGF-C also binds VEGFR-<em>2</em>, which is expressed in lymphatic endothelia, it is not clear which receptors are responsible for the lymphangiogenic effects of VEGF-C. VEGF-D, which binds to the same receptors, has been reported to induce angiogenesis, but its lymphangiogenic potential is not known. In order to define the lymphangiogenic signalling pathway we have created transgenic mice overexpressing a VEGFR-3-specific mutant of VEGF-C (VEGF-C156S) or VEGF-D in epidermal <em>keratinocytes</em> under the keratin 14 promoter. Both transgenes induced the <em>growth</em> of lymphatic vessels in the skin, whereas the blood vessel architecture was not affected. Evidence was also obtained that these <em>growth</em> <em>factors</em> act in a paracrine manner in vivo. These results demonstrate that stimulation of the VEGFR-3 signal transduction pathway is sufficient to induce specifically lymphangiogenesis in vivo.

We previously implicated tumor necrosis <em>factor</em>-alpha converting enzyme (TACE/ADAM17) in the processing of the integral membrane precursor to soluble transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha), pro-TGF-alpha. Here we examined TGF-alpha processing in a physiologically relevant cell model, primary <em>keratinocytes</em>, showing that cells lacking TACE activity shed dramatically less TGF-alpha as compared with wild-type cultures and that TGF-alpha cleavage was partially restored by infection of TACE-deficient cells with TACE-encoding adenovirus. Moreover, cotransfection of TACE-deficient fibroblasts with pro-TGF-alpha and TACE cDNAs increased shedding of mature TGF-alpha with concomitant conversion of cell-associated pro-TGF-alpha to a processed form. Purified TACE accurately cleaved pro-TGF-alpha in vitro at the N-terminal site and also cleaved a soluble form of pro-TGF-alpha containing only the ectodomain at the C-terminal site. In vitro, TACE accurately cleaved peptides corresponding to cleavage sites of several epidermal <em>growth</em> <em>factor</em> (EGF) family members, and transfection of TACE into TACE-deficient cells increased the shedding of amphiregulin and heparin-binding EGF (HB-EGF) proteins. Consistent with the hypothesis that TACE regulates EGF receptor (EGFR) ligand availability in vivo, mice heterozygous for Tace and homozygous for an impaired EGFR allele (wa-<em>2</em>) were born with open eyes significantly more often than Tace(+/+)Egfr(wa-<em>2</em>)(/)(wa-<em>2</em>) counterparts. Collectively, these data support a broad role for TACE in the regulated shedding of EGFR ligands.

The I kappa B kinase (IKK), consisting of the IKK1 and IKK<em>2</em> catalytic subunits and the NEMO (also known as IKK gamma) regulatory subunit, phosphorylates I kappa B proteins, targeting them for degradation and thus inducing activation of NF-kappa B (reviewed in refs 1, <em>2</em>). IKK<em>2</em> and NEMO are necessary for NF-kappa B activation through pro-inflammatory signals. IKK1 seems to be dispensable for this function but controls epidermal differentiation independently of NF-kappa B. Previous studies suggested that NF-kappa B has a function in the <em>growth</em> regulation of epidermal <em>keratinocytes</em>. Mice lacking RelB or I kappa B alpha, as well as both mice and humans with heterozygous NEMO mutations, develop skin lesions. However, the function of NF-kappa B in the epidermis remains unclear. Here we used Cre/loxP-mediated gene targeting to investigate the function of IKK<em>2</em> specifically in epidermal <em>keratinocytes</em>. IKK<em>2</em> deficiency inhibits NF-kappa B activation, but does not lead to cell-autonomous hyperproliferation or impaired differentiation of <em>keratinocytes</em>. Mice with epidermis-specific deletion of IKK<em>2</em> develop a severe inflammatory skin disease, which is caused by a tumour necrosis <em>factor</em>-mediated, alpha beta T-cell-independent inflammatory response that develops in the skin shortly after birth. Our results suggest that the critical function of IKK<em>2</em>-mediated NF-kappa B activity in epidermal <em>keratinocytes</em> is to regulate mechanisms that maintain the immune homeostasis of the skin.

Free radicals have been implicated in over a hundred disease conditions in humans, including arthritis, hemorrhagic shock, atherosclerosis, advancing age, ischemia and reperfusion injury of many organs, Alzheimer and Parkinson's disease, gastrointestinal dysfunctions, tumor promotion and carcinogenesis, and AIDS. Antioxidants are potent scavengers of free radicals and serve as inhibitors of neoplastic processes. A large number of synthetic and natural antioxidants have been demonstrated to induce beneficial effects on human health and disease prevention. However, the structure-activity relationship, bioavailability and therapeutic efficacy of the antioxidants differ extensively. Oligomeric proanthocyanidins, naturally occurring antioxidants widely available in fruits, vegetables, nuts, seeds, flowers and bark, have been reported to possess a broad spectrum of biological, pharmacological and therapeutic activities against free radicals and oxidative stress. We have assessed the concentration- or dose-dependent free radical scavenging ability of a novel IH636 grape seed proanthocyanidin extract (GSPE) both in vitro and in vivo models, and compared the free radical scavenging ability of GSPE with vitamins C, E and beta-carotene. These experiments demonstrated that GSPE is highly bioavailable and provides significantly greater protection against free radicals and free radical-induced lipid peroxidation and DNA damage than vitamins C, E and beta-carotene. GSPE was also shown to demonstrate cytotoxicity towards human breast, lung and gastric adenocarcinoma cells, while enhancing the <em>growth</em> and viability of normal human gastric mucosal cells. The comparative protective effects of GSPE, vitamins C and E were examined on tobacco-induced oxidative stress and apoptotic cell death in human oral <em>keratinocytes</em>. Oxidative tissue damage was determined by lipid peroxidation and DNA fragmentation, while apoptotic cell death was assessed by flow cytometry. GSPE provided significantly better protection as compared to vitamins C and E, singly and in combination. GSPE also demonstrated excellent protection against acetaminophen overdose-induced liver and kidney damage by regulating bcl-X(L) gene, DNA damage and presumably by reducing oxidative stress. GSPE demonstrated excellent protection against myocardial ischemia-reperfusion injury and myocardial infarction in rats. GSPE was also shown to upregulate bcl(<em>2</em>) gene and downregulate the oncogene c-myc. Topical application of GSPE enhances sun protection <em>factor</em> in human volunteers, as well as supplementation of GSPE ameliorates chronic pancreatitis in humans. These results demonstrate that GSPE provides excellent protection against oxidative stress and free radical-mediated tissue injury.

The hepatocyte nuclear <em>factor</em> 3alpha (HNF-3alpha) and 3beta proteins have homology in the winged helix/fork head DNA binding domain and regulate cell-specific transcription in hepatocytes and in respiratory and intestinal epithelia. In this study, we describe two novel isoforms of the winged helix transcription <em>factor</em> family, HNF-3/fork head homolog 11A (HFH-11A) and HFH-11B, isolated from the human colon carcinoma HT-<em>2</em>9 cell line. We show that these isoforms arise via differential splicing and are expressed in a number of epithelial cell lines derived from tumors (HT-<em>2</em>9, Caco-<em>2</em>, HepG<em>2</em>, HeLa, A549, and H441). We demonstrate that differentiation of Caco-<em>2</em> cells toward the enterocyte lineage results in decreased HFH-11 expression and reciprocal increases in HNF-3alpha and HNF-3beta mRNA levels. In situ hybridization of 16 day postcoitus mouse embryos demonstrates that HFH-11 expression is found in the mesenchymal and epithelial cells of the liver, lung, intestine, renal cortex, and urinary tract. Although HFH-11 exhibits a wide cellular expression pattern in the embryo, its adult expression pattern is restricted to epithelial cells of Lieberkühn's crypts of the intestine, the spermatocytes and spermatids of the testis, and the thymus and colon. HFH-11 expression is absent in adult hepatocytes, but its expression is reactivated in proliferating hepatocytes at 4, <em>2</em>4, and 48 h after partial hepatectomy. Consistent with these findings, we demonstrate that HFH-11 mRNA levels are stimulated by intratracheal administration of <em>keratinocyte</em> <em>growth</em> <em>factor</em> in adult lung and its expression in an adult endothelial cell line is reactivated in response to oxidative stress. These experiments show that the HFH-11 transcription <em>factor</em> is expressed in embryonic mesenchymal and epithelial cells and its expression is reactivated in these adult cell types by proliferative signals or oxidative stress.

Vascular endothelial <em>growth</em> <em>factor</em> (VEGF) has been implicated in the pathologic angiogenesis observed in psoriasis and other chronic inflammatory skin diseases that are characterized by enhanced expression of VEGF by epidermal <em>keratinocytes</em> and of VEGF receptors by tortuous microvessels in the upper dermis. To investigate the functional importance of chronic VEGF overexpression in vivo, we used a keratin 14 promoter expression cassette containing the gene for murine VEGF164 to selectively target VEGF expression to basal epidermal <em>keratinocytes</em> in transgenic mice. These mice demonstrated an increased density of tortuous cutaneous blood capillaries with elevated expression levels of the high affinity VEGF receptors, VEGFR-1 and VEGFR-<em>2</em>, most prominently during the neonatal period. In contrast, no abnormalities of lymphatic vessels were detected. In addition, the number of mast cells in the upper dermis was significantly increased in transgenic skin. Intravital fluorescence microscopy revealed highly increased leukocyte rolling and adhesion in postcapillary skin venules that were both inhibited after injection of blocking antibodies against E- and P-selectin. Combined blocking antibodies against intercellular adhesion molecule-1 and lymphocyte function-associated antigen-1 were without effect, whereas an anti-vascular cell adhesion molecule-1/VLA-4 antibody combination almost completely normalized the enhanced leukocyte adhesion in transgenic mice. This study reveals VEGF as a <em>growth</em> <em>factor</em> specific for blood vessels, but not lymphatic vessels, and demonstrates that chronic orthotopic overexpression of VEGF in the epidermis is sufficient to induce cardinal features of chronic skin inflammation, providing a molecular link between angiogenesis, mast cell accumulation, and leukocyte recruitment to sites of inflammation.

It is generally accepted that DNA damage and subsequent induction of apoptosis may be the primary cytotoxic mechanism of cisplatin and other DNA-binding antitumor drugs (Fisher,1994). Because the final step of apoptosis is characterized by morphological changes in the nucleus, the death signals of the execution phase must be transmitted from the cytoplasm to the nucleus. Thus, the recognition and processing of cisplatin-induced DNA damage through"classic" apoptosis, requires that a nuclear signal, generated at the initiation phase, be transmitted to the cytoplasm to be processed through the effector and execution phases. At the end of the execution phase, the apoptotic signal must come back to the nucleus to produce internucleosomal DNA degradation. Therefore, the induction of apoptosis from detection and subsequent processing of cisplatin-induced DNA damage seems to be a long and complex process of cell death. However, because cisplatin is a nonspecific drug and reacts not only with DNA but also with proteins,we cannot rule out the possibility that in some cases of cisplatin-induced apoptosis, an easier process of initiation, such as damage to cytoplasmic proteins, may take place (Pérez, 1998). Thus, damage to proteins is worth considering as a <em>factor</em> contributing to cisplatin-induced apoptosis. Moreover, it is possible that cisplatin damage to proteins could induce apoptosis at the execution phase level. In fact, initiation of apoptosis at the execution phase (activation of caspases) has been previously reported for the cell killing produced by cytotoxic T lymphocytes (Golstein et al., 1991). Although apoptosis and necrosis are conceptually distinct forms of cell death with very different morphological and biochemical characteristics, these two types of demise may occur simultaneously in tissues or cell cultures exposed to the same insult (Eguchi et al., 1997, Zhan et al., 1999). In fact, both types of cell death have been found in the same population of cisplatin-treated cells (Pestell et al., <em>2</em>000). Moreover, it has been hypothesized that in a tissue or cell population,apoptosis and necrosis might be two extremes of a continuum of possible types of cell demise. Individual cell death would be decided by <em>factors</em> such as the availability of energy and the metabolic condition of the cell (Leist et al., 1997). Thus, some cells might die as a result of an unfinished apoptotic program. In fact, in L1<em>2</em>10 leukemic cells, cisplatin-induced cell death seems to be the result of a defective apoptotic program that lacks some morphological and biochemical characteristics attributed to apoptosis (Segal-Bendirdjian and Jacquemin-Sablon, 1995). In addition, at high doses, cisplatin could damage molecules involved in cellular energy supply (i.e., ATP) and also proteins directly or indirectly involved in the apoptotic process (i.e., p53, Bax, Bcl-<em>2</em>, and caspases), leading to necrotic cell death. In fact, in cisplatin-resistant <em>keratinocytes</em> transformed by H-ras oncogene, a high dose of cisplatin (31<em>2</em> microM) induces characteristic features of necrotic cell death(Pérez et al., 1999). Thus, depending on the level of cellular damage induced by cisplatin, necrosis could take place either directly or as a consequence of an unfinished apoptotic program. In summary, a <em>growing</em> body of evidence suggests that cisplatin-induced cell death does not always come from "classic"apoptosis. Depending on both cisplatin dose and cellular status, cisplatin may also induced cell death by a defective apoptotic program or even by necrosis. Elucidation of the conditions under which the apoptotic program induced by cisplatin as well as other antitumor drugs is totally or partially executed may have important implications for the outcome of cancer chemotherapy.

The gene for fibroblast <em>growth</em> <em>factor</em> receptor-<em>2</em> (FGFR<em>2</em>) encodes two splice variants designated here as <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGFR) and bek. Their ligand-binding specificity is markedly different due to mutually exclusive alternative splicing. We asked whether alternative exon usage, in addition to influencing receptor specificity, could be correlated with transcriptional localization. This problem was studied by in situ hybridization and PCR, using probes and primers specific for the alternative exons of FGFR<em>2</em>. Transcripts of both variants were detected in all three germ layers within the embryonic and the extraembryonic areas of the primitive-streak embryo. The overall level of KGFR expression surpassed that of bek. The localized expression of both variant receptors was, however, more diffuse in the gastrula than later during organogenesis, when KGFR transcripts were evident mainly in epithelia, whereas bek was present in the corresponding mesenchymes. Our findings show the following: (1) Expression of both FGFR<em>2</em> variants is concordant with their involvement in murine gastrulation. They may endow competence to multiple areas, which may be restricted by their more confined ligands. (<em>2</em>) KGFR and bek seem to have unique roles in the development of the skin and its derivatives, whereas bek is preferentially expressed during osteogenesis. The two variants share potential regions of trans regulation in the genome; hence, we suggest that alternative splicing is jointly responsible for ligand binding and spatial specificity. (3) Finally, we defined the binding specificity of KGFR and bek to various FGF. The possibility of identifying specific functional areas for certain ligand-receptor pairs is discussed.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), also known as fibroblast <em>growth</em> <em>factor</em> 7 (FGF7), is synthesized by skin fibroblasts. However, its mitogenic activity is on skin <em>keratinocytes</em>, where it is the most potent <em>growth</em> <em>factor</em> identified thus far. To explore KGF's function in vivo, we used embryonic stem cell technology to generate mice lacking KGF. Over time, their fur developed a matted appearance, very similar to that of the rough mouse, whose recessive mutation maps at or near the KGF locus on mouse chromosome <em>2</em>. In contrast to the recently reported transforming <em>growth</em> <em>factor</em>-alpha (TGF-alpha) and FGF5 knockouts, which showed defects in the follicle outer-root sheath and the hair <em>growth</em> cycle, respectively, the hair defect in the KGF knockout seemed to be restricted to the cells giving rise to the hair shaft. Thus, we have uncovered a third, and at least partially nonoverlapping, <em>growth</em> <em>factor</em> pathway involved in orchestrating hair follicle <em>growth</em> and/or differentiation. Surprisingly, the absence of KGF resulted in no abnormalities in epidermal <em>growth</em> or wound healing. This was true even when we engineered double knockout mice, null for both KGF and TGF-alpha, two <em>factors</em> that are increased dramatically in the normal wound-healing process. Whereas we found no evidence of compensatory changes at the mRNA level of wounded knockout mice, these data imply that the regulation of epidermal <em>growth</em> is complex and involves a number of <em>growth</em> stimulatory <em>factors</em> that go beyond what are thought to be the major paracrine and autocrine <em>growth</em> <em>factors</em>. We suggest that the redundancy in epidermal <em>growth</em> and wound healing is likely to stem from the vitality of these functions to the organism, a feature that is not a consideration for the hair follicle.

Peroxiredoxin 6 (Prdx6) is the prototype and the only mammalian 1-Cys member of the Prdx family. Major differences from <em>2</em>-Cys Prdxs include the use of glutathione (GSH) instead of thioredoxin as the physiological reductant, heterodimerization with πGSH S-transferase as part of the catalytic cycle, and the ability either to reduce the oxidized sn-<em>2</em> fatty acyl group of phospholipids (peroxidase activity) or to hydrolyze the sn-<em>2</em> ester (alkyl) bond of phospholipids (phospholipase A(<em>2</em>) [PLA(<em>2</em>)] activity). The bifunctional protein has separate active sites for peroxidase (C47, R13<em>2</em>, H39) and PLA(<em>2</em>) (S3<em>2</em>, D140, H<em>2</em>6) activities. These activities are dependent on binding of the protein to phospholipids at acidic pH and to oxidized phospholipids at cytosolic pH. Prdx6 can be phosphorylated by MAP kinases at T177, which markedly increases its PLA(<em>2</em>) activity and broadens its pH-activity spectrum. Prdx6 is primarily cytosolic but also is targeted to acidic organelles (lysosomes, lamellar bodies) by a specific targeting sequence (amino acids 31-40). Oxidant stress and <em>keratinocyte</em> <em>growth</em> <em>factor</em> are potent regulators of Prdx6 gene expression. Prdx6 has important roles in both antioxidant defense based on its ability to reduce peroxidized membrane phospholipids and in phospholipid homeostasis based on its ability to generate lysophospholipid substrate for the remodeling pathway of phospholipid synthesis.

Recent studies have shown that application of basic fibroblast <em>growth</em> <em>factor</em> (basic FGF) to a wound has a beneficial effect. However, it has not been assessed whether endogenous FGF also plays a role in tissue repair. In this study we found a 160-fold induction of mRNA encoding <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) 1 day after skin injury. This large induction was unique within the family of FGFs, since mRNA levels of acidic FGF, basic FGF, and FGF-5 were only slightly induced (<em>2</em>- to 10-fold) during wound healing, and there was no expression of FGF-3, FGF-4, and FGF-6 detected in normal and wounded skin. High levels of FGF receptor 1 and FGF receptor <em>2</em> mRNA and low levels of FGF receptor 3 mRNA were found in both normal and wounded skin. No change in the levels of these transcripts was detected during wound healing. In situ hybridization studies revealed highest levels of KGF mRNA expression in the dermis at the wound edge and in the hypodermis below the wound. In contrast, mRNA encoding the receptor of this <em>growth</em> <em>factor</em> (a splice variant of FGF receptor <em>2</em>) was predominantly expressed in the epidermis. These results suggest that basal <em>keratinocytes</em> are stimulated by dermally derived KGF during wound healing and implicate a unique role of this member of the FGF family in wound repair.

p73 has been recently identified as a new structural and functional homologue of the transcription <em>factor</em> p53. It is expressed in either a full-length form, alpha, or a shorter beta mRNA variant, with exon 13 spliced out. Here we report the identification and functional characterization of two new p73 splicing variants, gamma (splicing out exon 11) and delta (splicing out exons 11, 1<em>2</em>, and 13). Both gamma and delta p73 variants are expressed in human peripheral blood lymphocytes, primary <em>keratinocytes</em>, and different tumor cell lines, including neuroblastoma, glioblastoma, melanoma, hepatoma, and leukemia. The expression pattern of the four p73 splicing variants differs in both primary cells of different lineage and established cell lines even within the same type of tumor. A two-hybrid assay was used to characterize the homodimeric and heterodimeric interactions between the p73 variants, and showed that neither p73gamma nor p73delta interact with p53, whereas p73gamma showed strong interactions with all p73 isoforms, and p73delta binds efficiently p73alpha and p73gamma but only weakly p73beta. At the functional level, p73gamma is significantly less efficient in activating transcription of the p<em>2</em>1(Waf1/Cip1) promoter than p53 or p73beta, whereas the effect of p73delta is intermediate and comparable to that of p73alpha. The ability of the different p73 variants to affect cell <em>growth</em> in p53 null osteosarcoma SAOS-<em>2</em> cells correlates with their transcriptional activity on the p<em>2</em>1(Waf1/Cip1) promoter: p73beta is the most efficient in inhibiting colony formation, whereas p73gamma is almost ineffective. Our results suggest that p73 isoforms may be differentially regulated, with four different isoforms capable of interacting among themselves and with p53. The relative expression level of each splice variant may modulate p73 transcriptional and <em>growth</em> suppression activities by affecting heterodimer formation.

OBJECTIVE

In gout, incompletely defined molecular <em>factors</em> alter recognition of dormant articular and bursal monosodium urate monohydrate (MSU) crystal deposits, thereby inducing self-limiting bouts of characteristically severe neutrophilic inflammation. To define primary determinants of cellular recognition, uptake, and inflammatory responses to MSU crystals, we conducted a study to test the role of Toll-like receptor <em>2</em> (TLR-<em>2</em>), TLR-4, and the cytosolic TLR adapter protein myeloid differentiation <em>factor</em> 88 (MyD88), which are centrally involved in innate immune recognition of microbial pathogens.

METHODS

We isolated bone marrow-derived macrophages (BMDMs) in TLR-<em>2</em>-/-, TLR-4-/-, MyD88-/-, and congenic wild-type mice, and assessed phagocytosis and cytokine expression in response to endotoxin-free MSU crystals under serum-free conditions. MSU crystals also were injected into mouse synovium-like subcutaneous air pouches.

RESULTS

TLR-<em>2</em>-/-, TLR-4-/-, and MyD88-/- BMDMs demonstrated impaired uptake of MSU crystals in vitro. MSU crystal-induced production of interleukin-1beta (IL-1beta), tumor necrosis <em>factor</em> alpha, keratinocyte-derived cytokine/growth-related oncogene alpha, and transforming growth <em>factor</em> beta1 also were significantly suppressed in TLR-<em>2</em>-/- and TLR-4-/- BMDMs and were blunted in MyD88-/- BMDMs in vitro. Neutrophil influx and local induction of IL-1beta in subcutaneous air pouches were suppressed 6 hours after injection of MSU crystals in TLR-<em>2</em>-/- and TLR-4-/- mice and were attenuated in MyD88-/- mice.

CONCLUSIONS

The murine host requires TLR-<em>2</em>, TLR-4, and MyD88 for macrophage activation and development of full-blown neutrophilic, air pouch inflammation in response to MSU crystals. Our findings implicate innate immune cellular recognition of naked MSU crystals by specific TLRs as a major <em>factor</em> in determining the inflammatory potential of MSU crystal deposits and the course of gouty arthritis.

Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) inhibits cell cycle progression, in part through up-regulation of gene expression of the p<em>2</em>1(WAF1/Cip1) (p<em>2</em>1) cell cycle inhibitor. Previously we have reported that the intracellular effectors of TGF-beta, Smad3 and Smad4, functionally cooperate with Sp1 to activate the human p<em>2</em>1 promoter in hepatoma HepG<em>2</em> cells. In this study we show that Smad3 and Smad4 when overexpressed in HaCaT <em>keratinocytes</em> lead to activation of the p<em>2</em>1 promoter. Activation requires the binding sites for the ubiquitous transcription <em>factor</em> Sp1 on the proximal promoter. Induction of the endogenous HaCaT p<em>2</em>1 gene by TGF-beta1 is further enhanced after overexpression of Smad3 and Smad4, whereas dominant negative mutants of Smad3 and Smad4 and the inhibitory Smad7 all inhibit p<em>2</em>1 induction by TGF-beta1 in a dose-dependent manner. We show that Sp1 expressed in the Sp1-deficient Drosophila SL-<em>2</em> cells binds to the proximal p<em>2</em>1 promoter sequences, whereas Smad proteins do not. In support of this finding, we show that DNA-binding domain mutants of Smad3 and Smad4 are capable of transactivating the p<em>2</em>1 promoter as efficiently as wild type Smads. Co-expression of Smad3 with Smad4 and Sp1 in SL-<em>2</em> cells or co-incubation of phosphorylated Smad3, Smad4, and Sp1 in vitro results in enhanced binding of Sp1 to the p<em>2</em>1 proximal promoter sequences. We demonstrate that Sp1 physically and directly interacts with Smad<em>2</em>, Smad3, and weakly with Smad4 via their amino-terminal (Mad-Homology 1) domain. Finally, by using GAL4 fusion proteins we show that the glutamine-rich sequences in the transactivation domain of Sp1 contribute to the cooperativity with Smad proteins. In conclusion, Smad proteins play important roles in regulation of the p<em>2</em>1 gene by TGF-beta, and the functional cooperation of Smad proteins with Sp1 involves the physical interaction of these two types of transcription <em>factors</em>.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is a potent mitogen for epithelial cells, and it promotes survival of these cells under stress conditions. In a search for KGF-regulated genes in <em>keratinocytes</em>, we identified the gene encoding the transcription <em>factor</em> NF-E<em>2</em>-related <em>factor</em> <em>2</em> (Nrf<em>2</em>). Nrf<em>2</em> is a key player in the cellular stress response. This might be of particular importance during wound healing, where large amounts of reactive oxygen species are produced as a defense against invading bacteria. Therefore, we studied the wound repair process in Nrf<em>2</em> knockout mice. Interestingly, the expression of various key players involved in wound healing was significantly reduced in early wounds of the Nrf<em>2</em> knockout animals, and the late phase of repair was characterized by prolonged inflammation. However, these differences in gene expression were not reflected by obvious histological abnormalities. The normal healing rate appears to be at least partially due to an up-regulation of the related transcription <em>factor</em> Nrf3, which was also identified as a target of KGF and which was coexpressed with Nrf<em>2</em> in the healing skin wound. Taken together, our results reveal novel roles of the KGF-regulated transcription <em>factors</em> Nrf<em>2</em> and possibly Nrf3 in the control of gene expression and inflammation during cutaneous wound repair.

One important action of <em>growth</em> <em>factors</em> is their participation in tissue repair; however, the signaling pathways involved are poorly understood. In a model of corneal wound healing, we found that two paracrine <em>growth</em> <em>factors</em>, hepatocyte <em>growth</em> <em>factor</em> (HGF) and <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), induced rapid and marked activation and prompt nuclear accumulation of phospho-p38 (p-p38) and -ERK1/<em>2</em> (p-ERK1/<em>2</em>), but not of JNK (p-JNK1/<em>2</em>), in corneal epithelial cells. Interruption of p38 and ERK1/<em>2</em> signaling pathways by pretreatment with inhibitors SB<em>2</em>03580 and PD98059 and subsequent stimulation with HGF or KGF abolished the activation and nuclear localization. Inhibition of either one of these mitogen-activated protein kinases, p38 or ERK1/<em>2</em>, induced a robust cross-activation of the other. In immunofluorescence studies of wounded cornea, p-p38, unlike p-ERK1/<em>2</em>, was immediately detectable in epithelium after injury. Inhibition of p38 by SB<em>2</em>03580 blocked migration of epithelial cells almost completely. In contrast, PD98059 seemed to slightly increase the migration, through concomitant activation of p38. Unlike ERK1/<em>2</em>, p38 did not significantly contribute to proliferation of epithelial cells. Inhibition of either the ERK1/<em>2</em> or p38 pathway resulted in delayed corneal epithelial wound healing. Interruption of both signaling cascades additively inhibited the wound-healing process. These findings demonstrate that both p38 and ERK1/<em>2</em> coordinate the dynamics of wound healing: while <em>growth</em> <em>factor</em>-stimulated p38 induces epithelial migration, ERK1/<em>2</em> activation induces proliferation. The cross-talk between these two signal cascades and the selective action of p38 in migration appear to be important to corneal wound healing, and possibly wound healing in general, and may offer novel drug targets for tissue repair.

Wound fluid was obtained from porcine partial-thickness excisional wounds and analyzed for heparin-binding <em>growth</em> <em>factors</em>. Two heparin-binding <em>growth</em> <em>factor</em> activities were detected, a relatively minor one that was eluted from a heparin affinity column with 0.65 M NaCl and a major one that was eluted with 1.1 M NaCl. These activities were not present in wound fluid 1 hr after injury but appeared 1 day after injury, were maximal <em>2</em>-3 days after injury, and were not detectable by 8 days after injury. The heparin-binding <em>growth</em> <em>factor</em> eluted with 0.65 M NaCl was identified as a platelet-derived <em>growth</em> <em>factor</em> (PDGF)-like activity by the use of specific anti-PDGF neutralizing antibodies. The heparin-binding <em>growth</em> <em>factor</em> eluted with 1.1 M NaCl was shown to be structurally related to heparin-binding epidermal <em>growth</em> <em>factor</em> (EGF)-like <em>growth</em> <em>factor</em> (HB-EGF) by several criteria, including binding to heparin affinity columns and elution with 1.1 M NaCl, competition with the binding of 1<em>2</em>5I-EGF to the EGF receptor, triggering phosphorylation of the EGF receptor, immunodetection on a Western blot, and stimulation of fibroblast and <em>keratinocyte</em> <em>growth</em>. It was concluded that HB-EGF is a major <em>growth</em> <em>factor</em> component of wound fluid and, since it is mitogenic for fibroblasts and <em>keratinocytes</em>, that it might play an important role in wound healing.

The importance of angiogenesis in malignant tumor <em>growth</em> has been interpreted mainly in terms of oxygen and nutrient supply. Here we demonstrate its fundamental role for tumor invasion of malignant human <em>keratinocytes</em> in surface transplants on nude mice. Distinct patterns of angiogenesis and vascular endothelial <em>growth</em> <em>factor</em> receptor-<em>2</em> (VEGFR-<em>2</em>) expression allowed us to distinguish between benign and malignant cells. Functional inactivation of VEGF-R<em>2</em> by a blocking antibody disrupted ongoing angiogenesis and prevented invasion of malignant cells, without reducing tumor cell proliferation. The reversion of a malignant into a benign phenotype by halting angiogenesis demonstrates a significant function of vascular endothelium for tumor invasion.