Stimulation of <em>growth</em> and differentiation of human epidermis by epidermal <em>growth</em> <em>factor</em> (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm<em>2</em> punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal <em>keratinocytes</em> were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a <em>2</em>-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [1<em>2</em>5I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal <em>growth</em> <em>factor</em> was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers.

ADAM17 (a disintegrin and metalloproteinase 17) is ubiquitously expressed and cleaves membrane proteins, such as epidermal <em>growth</em> <em>factor</em> receptor (EGFR) ligands, l-selectin, and TNF, from the cell surface, thus regulating responses to tissue injury and inflammation. However, little is currently known about its role in skin homeostasis. We show that mice lacking ADAM17 in <em>keratinocytes</em> (A17(ΔKC)) have a normal epidermal barrier and skin architecture at birth but develop pronounced defects in epidermal barrier integrity soon after birth and develop chronic dermatitis as adults. The dysregulated expression of epidermal differentiation proteins becomes evident <em>2</em> d after birth, followed by reduced transglutaminase (TGM) activity, transepidermal water loss, up-regulation of the proinflammatory cytokine IL-36α, and inflammatory immune cell infiltration. Activation of the EGFR was strongly reduced in A17(ΔKC) skin, and topical treatment of A17(ΔKC) mice with recombinant TGF-α significantly improved TGM activity and decreased skin inflammation. Finally, we show that mice lacking the EGFR in <em>keratinocytes</em> (Egfr(ΔKC)) closely resembled A17(ΔKC) mice. Collectively, these results identify a previously unappreciated critical role of the ADAM17-EGFR signaling axis in maintaining the homeostasis of the postnatal epidermal barrier and suggest that this pathway could represent a good target for treatment of epidermal barrier defects.

Although there is experimental evidence supporting the involvement of hepatic stem cells in the pathogenesis of liver cancers, the detection and isolation of these cells remains elusive. A logical approach to detecting these cells would take advantage of their ability to differentiate (or to give rise to cells that differentiate) into hepatocytes. This approach requires an assay system that is conducive to hepatocytic differentiation. Here, we report the development of an in vitro system consisting of a three-dimensional collagen gel matrix and a fibroblast feeder layer that supports hepatocytic differentiation from precursor epithelial (oval) cell lines. The LE/<em>2</em> and LE/6 oval cell lines used in this study are nontumorigenic cells that are derived from the livers of adult rats fed a choline-deficient diet containing 0.1% ethionine for <em>2</em> and 6 weeks, respectively. These lines consist of small cells that are phenotypically immature with few cytoplasmic organelles and a high nuclear-to-cytoplasmic ratio. After 4 weeks in the three-dimensional culture system, these cells acquired typical hepatocytic morphology. By electron microscopy, the cells formed canalicular structures that are typical of hepatocytes and were organelle rich, displaying peroxisomes, abundant mitochondria, and rough endoplasmic reticulum. The cells produced albumin and displayed a cytokeratin (CK) pattern typical of hepatocytes (CK 8 and CK 18-positive and CK 19-negative). The presence of a mesenchymal cell feeder layer was essential for supporting hepatocytic differentiation. Without a feeder layer but in the presence of hepatocyte <em>growth</em> <em>factor</em> and/or <em>keratinocyte</em> <em>growth</em> <em>factor</em>, the precursor cells formed ductal structures, suggestive of differentiation along the bile duct lineage. The three-dimensional system described provides direct proof of the lineage generation capacity of oval cells. It offers a model to study <em>factors</em> that may be important for hepatocytic differentiation from precursor cells and a means to assay cell populations for their ability to give rise to normal and transformed hepatocytes.

AMP-activated protein kinase or AMPK is an evolutionarily conserved sensor of cellular energy status, activated by a variety of cellular stresses that deplete ATP. However, the possible involvement of AMPK in UV- and H(<em>2</em>)O(<em>2</em>)-induced oxidative stresses that lead to skin aging or skin cancer has not been fully studied. We demonstrated for the first time that UV and H(<em>2</em>)O(<em>2</em>) induce AMPK activation (Thr(17<em>2</em>) phosphorylation) in cultured human skin <em>keratinocytes</em>. UV and H(<em>2</em>)O(<em>2</em>) also phosphorylate LKB1, an upstream signal of AMPK, in an epidermal <em>growth</em> <em>factor</em> receptor-dependent manner. Using compound C, a specific inhibitor of AMPK and AMPK-specific small interfering RNA knockdown as well as AMPK activator, we found that AMPK serves as a positive regulator for p38 and p53 (Ser(15)) phosphorylation induced by UV radiation and H(<em>2</em>)O(<em>2</em>) treatment. We also observed that AMPK serves as a negative feedback signal against UV-induced mTOR (mammalian target of rapamycin) activation in a TSC<em>2</em>-dependent manner. Inhibiting mTOR and positively regulating p53 and p38 might contribute to the pro-apoptotic effect of AMPK on UV- or H(<em>2</em>)O(<em>2</em>)-treated cells. Furthermore, activation of AMPK also phosphorylates acetyl-CoA carboxylase or ACC, the pivotal enzyme of fatty acid synthesis, and PFK<em>2</em>, the key protein of glycolysis in UV-radiated cells. Collectively, we conclude that AMPK contributes to UV- and H(<em>2</em>)O(<em>2</em>)-induced apoptosis via multiple mechanisms in human skin <em>keratinocytes</em> and AMPK plays important roles in UV-induced signal transduction ultimately leading to skin photoaging and even skin cancer.

The K-sam gene, originally isolated as an amplified gene from the stomach cancer cell line KATO-III, is characterized by its preferential amplification in the undifferentiated type (diffuse type) of stomach cancer and encodes one of the receptors for heparin-binding <em>growth</em> <em>factors</em> or fibroblast <em>growth</em> <em>factors</em>. The K-sam gene has been isolated by different methods and has been designated BEK, TK14, and Cek<em>2</em>. The receptor for <em>keratinocyte</em> <em>growth</em> <em>factor</em> was also found to be encoded by the same gene. To examine the expression of the K-sam protein in stomach cancer, polyclonal antibody pK1-<em>2</em> was raised against the extracellular domain of the gene product. This antibody detected K-sam proteins by Western blot and flow cytometry analyses in stomach cancer cell lines KATO-III and HSC39, in which the K-sam gene is amplified and overexpressed. By immunohistochemical analysis, <em>2</em>0 of 38 cases of the undifferentiated type of advanced stomach cancer were K-sam positive, whereas none of 11 cases of the differentiated or intestinal type revealed K-sam staining. The K-sam product was observed predominantly in diffusely infiltrative lesions. In one autopsy case, the K-sam protein was detected only focally in the primary tumor, whereas markedly increased staining for the K-sam product was detected diffusely in the metastasized tumor in the lymph node and liver. These results suggest that K-sam overexpression is associated with the malignant phenotype of the undifferentiated type of stomach cancer, such as infiltrative <em>growth</em> and metastasis.

The endothelial cell-specific mitogen vascular permeability <em>factor</em>/vascular endothelial <em>growth</em> <em>factor</em> (VPF/VEGF) represents a central regulator of cutaneous angiogenesis. Increased VPF/VEGF expression has recently been reported in psoriatic skin and healing wounds, both conditions in which transforming <em>growth</em> <em>factor</em>-alpha (TGF alpha) and its ligand, the epidermal <em>growth</em> <em>factor</em> receptor, are markedly up-regulated. Since TGF alpha strongly induces VPF/VEGF synthesis in <em>keratinocytes</em>, TGF alpha-mediated VPF/VEGF expression is likely to play a significant role in the initiation and maintenance of increased vascular hyperpermeability and hyperproliferation in skin biology. The objectives of the present studies were to determine the molecular mechanisms responsible for TGF alpha-induced transcriptional activation of the VPF/VEGF gene. We have identified a GC-rich TGF alpha-responsive region between -88 bp and -65 bp of the VPF/VEGF promoter that is necessary for constitutive and TGF alpha-inducible transcriptional activation. In electrophoretic mobility shift assays, this region binds Sp1-dependent protein complexes constitutively and an additional TGF alpha-inducible protein complex that is distinct from Sp1 protein. Both AP-<em>2</em> and Egr-1 transcription <em>factors</em> were detected as components of the TGF alpha-inducible protein complex in supershift EMSA studies. In co-transfection studies, an AP-<em>2</em> but not an Egr-1 expression vector activated VPF/VEGF transcription, thus indicating that AP-<em>2</em> protein is functionally important in TGF alpha-induced VPF/VEGF gene expression. By clarifying regulatory mechanisms that are critical for angiogenic processes in the skin, these studies may form the basis for new therapeutic strategies to modulate VPF/VEGF expression in cutaneous inflammation and wound healing.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) activates <em>keratinocyte</em> migration and stimulates wound healing. Hyaluronan, an extracellular matrix glycosaminoglycan that accumulates in wounded epidermis, is known to promote cell migration, suggesting that increased synthesis of hyaluronan might be associated with the KGF response in <em>keratinocytes</em>. Treatment of monolayer cultures of rat epidermal <em>keratinocytes</em> led to an elongated and lifted cell shape, increased filopodial protrusions, enhanced cell migration, accumulation of intermediate size hyaluronan in the culture medium and within <em>keratinocytes</em>, and a rapid increase of hyaluronan synthase <em>2</em> (Has<em>2</em>) mRNA, suggesting a direct influence on this gene. In stratified, organotypic cultures of the same cell line, both Has<em>2</em> and Has3 with the hyaluronan receptor CD44 were up-regulated and hyaluronan accumulated in the epidermis, the spinous cell layer in particular. At the same time the expression of the early differentiation marker keratin 10 was inhibited, whereas filaggrin expression and epidermal permeability were less affected. The data indicate that Has<em>2</em> and Has3 belong to the targets of KGF in <em>keratinocytes</em>, and support the idea that enhanced hyaluronan synthesis acts an effector for the migratory response of <em>keratinocytes</em> in wound healing, whereas it may delay <em>keratinocyte</em> terminal differentiation.

OBJECTIVE

AZD2171 is an oral, highly potent, and selective vascular endothelial growth factor signaling inhibitor that inhibits all vascular endothelial growth factor receptor tyrosine kinases. The purpose of this study was to investigate the activity of AZD2171 in gastric cancer.

METHODS

We examined the antitumor effect of AZD2171 on the eight gastric cancer cell lines in vitro and in vivo.

RESULTS

AZD2171 directly inhibited the growth of two gastric cancer cell lines (KATO-III and OCUM2M), with an IC(50) of 0.15 and 0.37 micromol/L, respectively, more potently than the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib. Reverse transcription-PCR experiments and immunoblotting revealed that sensitive cell lines dominantly expressed COOH terminus-truncated fibroblast growth factor receptor 2 (FGFR2) splicing variants that were constitutively phosphorylated and spontaneously dimerized. AZD2171 completely inhibited the phosphorylation of FGFR2 and downstream signaling proteins (FRS2, AKT, and mitogen-activated protein kinase) in sensitive cell lines at a 10-fold lower concentration (0.1 micromol/L) than in the other cell lines. An in vitro kinase assay showed that AZD2171 inhibited kinase activity of immunoprecipitated FGFR2 with submicromolar K(i) values ( approximately 0.05 micromol/L). Finally, we assessed the antitumor activity of AZD2171 in human gastric tumor xenograft models in mice. Oral administration of AZD2171 (1.5 or 6 mg/kg/d) significantly and dose-dependently inhibited tumor growth in mice bearing KATO-III and OCUM2M tumor xenografts.

CONCLUSIONS

AZD2171 exerted potent antitumor activity against gastric cancer xenografts overexpressing FGFR2. The results of these preclinical studies indicate that AZD2171 may provide clinical benefit in patients with certain types of gastric cancer.

The major obstacles to successful outcome after allogeneic bone marrow transplantation (BMT) for leukemia remain graft-versus-host disease (GVHD) and leukemic relapse. Improved survival after BMT therefore requires more effective GVHD prophylaxis that does not impair graft-versus-leukemia (GVL) effects. We studied the administration of human recombinant <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) in a well- characterized murine BMT model for its effects on GVHD. KGF administration from day -3 to +7 significantly reduced GVHD mortality and the severity of GVHD in the gastrointestinal (GI) tract, reducing serum lipopolysaccharide (LPS) and tumor necrosis <em>factor</em> (TNF)alpha levels, but preserving donor T-cell responses (cytotoxic T lymphocyte [CTL] activity, proliferation, and interleukin [IL]-<em>2</em> production) to host antigens. When mice received lethal doses of P815 leukemia cells at the time of BMT, KGF treatment significantly decreased acute GVHD compared with control-treated allogeneic mice and resulted in a significantly improved leukemia-free survival (4<em>2</em>% v 4%, P <.001). KGF administration thus offers a novel approach to the separation of GVL effects from GVHD.

To address the functions of Rac1 in <em>keratinocytes</em> of the basal epidermal layer and in the outer root sheath of hair follicles, we generated transgenic mice expressing a dominant inhibitory mutant of Rac, N17Rac1, under the control of the keratin 14 promoter. These mice do not exhibit an overt skin phenotype but show protracted skin wound re-epithelialization. Investigation into the underlying mechanisms revealed that in vivo both proliferation of wound-edge <em>keratinocytes</em> and centripetal migration of the neo-epidermis were impaired. Similar results were obtained in mice with an epidermis-specific deletion of Rac1. Primary epidermal <em>keratinocytes</em> that expressed the N17Rac1 transgene were less proliferative than control cells and showed reduced ERK1/<em>2</em> phosphorylation upon <em>growth</em> <em>factor</em> stimulation. Adhesion, spreading, random migration and closure of scratch wounds in vitro were significantly inhibited on collagen I and, to a lesser extent, on fibronectin. Stroboscopic analysis of cell dynamics (SACED) of N17Rac1 transgenic and control <em>keratinocytes</em> identified decreased lamella-protrusion persistence in connection with increased ruffle frequency as a probable mechanism for the observed impairment of <em>keratinocyte</em> adhesion and migration. We conclude that Rac1 is functionally required for normal epidermal wound healing and, in this context, exerts a dual function - namely the regulation of <em>keratinocyte</em> proliferation and migration.

Melanocytes characterized by the activities of tyrosinase, tyrosinase-related protein (TRP)-1 and TRP-<em>2</em> as well as by melanosomes and dendrites are located mainly in the epidermis, dermis and hair bulb of the mammalian skin. Melanocytes differentiate from melanoblasts, undifferentiated precursors, derived from embryonic neural crest cells. Because hair bulb melanocytes are derived from epidermal melanoblasts and melanocytes, the mechanism of the regulation of the proliferation and differentiation of epidermal melanocytes should be clarified. The regulation by the tissue environment, especially by <em>keratinocytes</em> is indispensable in addition to the regulation by genetic <em>factors</em> in melanocytes. Recent advances in the techniques of tissue culture and biochemistry have enabled us to clarify <em>factors</em> derived from <em>keratinocytes</em>. Alpha-melanocyte-stimulating hormone, adrenocorticotrophic hormone, basic fibroblast <em>growth</em> <em>factor</em>, nerve <em>growth</em> <em>factor</em>, endothelins, granulocyte-macrophage colony-stimulating <em>factor</em>, steel <em>factor</em>, leukemia inhibitory <em>factor</em> and hepatocyte <em>growth</em> <em>factor</em> have been suggested to be the <em>keratinocyte</em>-derived <em>factors</em> and to regulate the proliferation and/or differentiation of mammalian epidermal melanocytes. Numerous <em>factors</em> may be produced in and released from <em>keratinocytes</em> and be involved in regulating the proliferation and differentiation of mammalian epidermal melanocytes through receptor-mediated signaling pathways.

Reiterative in vitro selection-amplification from random oligonucleotide libraries allows the identification of molecules with specific functions such as binding to specific proteins. The therapeutic usefulness of such molecules depends on their high affinity and nuclease resistance. Libraries of RNA molecules containing <em>2</em>'amino-(<em>2</em>'NH<em>2</em>)- or <em>2</em>'fluoro-(<em>2</em>'F)-<em>2</em>'-deoxypyrimidines could yield ligands with similar nuclease resistance but not necessarily with similar affinities. This is because the intramolecular helices containing <em>2</em>'NH<em>2</em> have lower melting temperatures (Tm) compared with helices containing <em>2</em>'F, giving them thermodynamically less stable structures and possibly weaker affinities. We tested these ideas by isolating high-affinity ligands to human <em>keratinocyte</em> <em>growth</em> <em>factor</em> from libraries containing modified RNA molecules with either <em>2</em>'NH<em>2</em> or <em>2</em>'F pyrimidines. We demonstrated that <em>2</em>'F RNA ligands have affinities (Kd approximately 0.3-3 pM) and bioactivities (Ki approximately 34 pM) superior to <em>2</em>'NH<em>2</em> ligands (Kd approximately 400 pM and Ki approximately 10 nM). In addition, <em>2</em>'F ligands have extreme thermo-stabilities (Tm approximately 78 degrees C in low salt, and specificities).

Dioxin (<em>2</em>,3,7,8-tetrachlorodibenzo-p-dioxin, TCDD), a widespread environmental contaminant, may elicit its effects by altering gene expression in susceptible cells. Five TCDD-responsive complementary DNA clones were isolated from a human <em>keratinocyte</em> cell line. One of these clones encodes plasminogen activator inhibitor-<em>2</em>, a <em>factor</em> that influences <em>growth</em> and differentiation by regulating proteolysis of the extracellular matrix. Another encodes the cytokine interleukin-1 beta. Thus, TCDD alters the expression of <em>growth</em> regulatory genes and has effects similar to those of other tumor-promoting agents that affect both inflammation and differentiation.

CCAAT-enhancer binding proteins (C/EBP) are basic region/leucine zipper (bZIP) transcription <em>factors</em> selectively expressed during the differentiation of liver, adipose tissue, blood cells, and the endocrine pancreas. Here we show that C/EBP isoforms are differentially expressed in the skin. BALB/MK <em>keratinocytes</em> incubated in 0.1<em>2</em> mM calcium medium undergo a differentiation program featuring <em>growth</em>-arrest at <em>2</em>4-48 h, keratin K10 gene expression beginning at <em>2</em>4 h, and apoptosis commencing at 48 h. Within this framework, western immunoblot analysis and immunohistochemistry reveal that C/EBP alpha increases 5-fold at 1-<em>2</em> d and remains elevated, C/EBP beta rises <em>2</em>-fold at <em>2</em>-4 d and gradually falls, and CHOP rises 9-fold in the first <em>2</em>4 h then returns rapidly to baseline. Several products of alternative translation are observed in BALB/MK cells, i.e., 4<em>2</em> kDa and 30 kDa forms of C/EBP alpha, and 3<em>2</em> kDa and <em>2</em>0 kDa forms of C/EBP beta. By immunohistologic examination of human, rat, and mouse skin, all three transcription <em>factors</em> are highly expressed within epithelial compartments in a spatially restricted distribution. C/EBP alpha is concentrated in the upper epidermis in a predominantly cytoplasmic location within cells, whereas the highest levels of C/EBP beta and CHOP are seen in the mid-epidermis, mainly within nuclei. High levels of C/EBP beta and CHOP (but not C/EBP alpha) are also observed in hair follicles and sebaceous glands. The identity of these <em>factors</em> in the epidermis is confirmed by western immunoblot analyses. In summary, C/EBP are expressed in a differentiation-associated manner in the skin, and may play an important role in regulating one or more aspects of the epidermal differentiation program.

Adipose-derived stem cells (ADSCs) and their secretomes mediate diverse skin-regeneration effects, such as wound-healing and antioxidant protection, that are enhanced by hypoxia. We investigated the hair-<em>growth</em>-promoting effect of conditioned medium (CM) of ADSCs to determine if ADSCs and their secretomes regenerate hair and if hypoxia enhances hair regeneration. If so, we wanted to identify the <em>factors</em> responsible for hypoxia-enhanced hair-regeneration. We found that ADSC-CM administrated subcutaneously induced the anagen phase and increased hair regeneration in C(3)H/NeH mice. In addition, ADSC-CM increased the proliferation of human follicle dermal papilla cells (HFDPCs) and human epithelial <em>keratinocytes</em> (HEKs), which are derived from two major cell types present in hair follicles. We investigated the effect of hypoxia on ADSC function using the same animal model in which hypoxia increased hair re<em>growth</em>. Forty-one <em>growth</em> <em>factors</em> in ADSC-CM from cells cultured under hypoxic or normoxic conditions were analyzed. The secretion of insulin-like <em>growth</em> <em>factor</em> binding protein (IGFBP)-1, IGFBP-<em>2</em>, macrophage colony-stimulating <em>factor</em> (M-CSF), M-CSF receptor, platelet-derived <em>growth</em> <em>factor</em> receptor-beta, and vascular endothelial <em>growth</em> <em>factor</em> was significantly increased by hypoxia, while the secretion of epithelial <em>growth</em> <em>factor</em> production was decreased. It is reasonable to conclude that ADSCs promote hair <em>growth</em> via a paracrine mechanism that is enhanced by hypoxia.

Hair follicle development serves as an excellent model to study control of organ morphogenesis. Three specific isoforms of TGF-beta exist which exhibit a distinct pattern of expression during hair follicle morphogenesis. To clarify the still elusive role of these <em>factors</em> in hair follicle development, we have used a combined genetic and functional approach: analysis of hair follicle development in mice with disruptions of the TGF-beta1, <em>2</em>, and 3 genes was coupled with a direct functional test of the effect of added purified <em>factors</em> on fetal hair follicle development in skin organ cultures. TGF-beta<em>2</em> null mice exhibited a profound delay of hair follicle morphogenesis, with a 50% reduced number of hair follicles. In contrast to hair follicle development, <em>growth</em> and differentiation of interfollicular <em>keratinocytes</em> proceeded unimpaired. Unlike TGF-beta<em>2</em>-/- mice, mice with a disruption of the TGF-beta1 gene showed slightly advanced hair follicle formation, while lack of the TGF-beta3 gene did not have any effects. Treatment of wild-type, embryonic skin explants (E14.5 or E15.5) with TGF-beta<em>2</em> protein in either soluble form or slow release beads induced hair follicle development and epidermal hyperplasia, while similar TGF-beta1 treatment exerted suppressive effects. Thus, the TGF-beta<em>2</em> isoform plays a specific role, not shared by the other TGF-beta isoforms, as an inducer of hair follicle morphogenesis and is both required and sufficient to promote this process.

Wound healing is a complex biological process that requires cellular interactions between a variety of cells, including fibroblasts, myofibroblasts, smooth muscle cells, endothelial cells, <em>keratinocytes</em> and immune cells. These interactions are mediated by numerous <em>factors</em> such as <em>growth</em> <em>factors</em>, hormones, blood components and second messengers. Several <em>growth</em> <em>factors</em> that are released at the wound site are presumed to be necessary for wound healing. These include epidermal <em>growth</em> <em>factor</em> (EGF), fibroblast <em>growth</em> <em>factor</em> (FGF), insulin-like <em>growth</em> <em>factor</em> (IGF), <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), platelet-derived <em>growth</em> <em>factor</em> (PDGF), transforming <em>growth</em> <em>factor</em> (TGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF). The clinical use of <em>growth</em> <em>factors</em> to stimulate the healing of wounds is currently being investigated. Several <em>growth</em> <em>factors</em>, including PDGF, FGF-<em>2</em>, IGF and KGF, have been used in clinical trials, and PDGF is currently approved for use in human medicine.

OBJECTIVE

Angiogenesis is a process stimulated in inflamed synovium of patients with osteoarthritis (OA), and contributes to the progression of the disease. Synovial fibroblasts secrete angiogenic factors, such as vascular endothelial growth factor (VEGF), an up-regulator of angiogenesis, and this ability is increased by interleukin (IL)-1beta. The purpose of this study was to verify whether IL-17 contributes and/or synergizes with IL-1beta and tumor necrosis factor (TNF)-alpha in vessel development in articular tissues by stimulating the secretion of proangiogenic factors by synovial fibroblasts.

METHODS

We stimulated in vitro synovial fibroblasts isolated from OA, rheumatoid arthritis (RA) and fractured patients (FP) with IL-17 and IL-1beta and from OA patients with IL-17, IL-1beta and TNF-alpha. In the supernatants from the cultures, we assayed the amount of VEGF by immunoassay and other angiogenic factors (keratinocyte growth factor, KGF; hepatocyte growth factor, HGF; heparin-binding endothelial growth factor, HB-EGF; angiopoietin-2, Ang-2; platelet-derived growth factor B, PDGF-BB; thrombopoietin, TPO) by chemiluminescence; semiquantitative RT-PCR was used to state mRNA expression of nonreleased angiogenic factors (Ang-2 and PDGF-BB) and tissue inhibitors of metalloproteinase (TIMP)-1.

RESULTS

IL-17, TNF-alpha and IL-1beta increased VEGF secretion by synovial fibroblasts from OA patients. IL-17 and IL-1beta also increased VEGF secretion in RA and FP. Besides, IL-17 increased KGF and HGF secretions in OA, RA and FP; in OA and RA, IL-17 also increased the HB-EGF secretion and the expression of TIMP-1 as protein and mRNA. In OA patients IL-17 had an additive effect on TNF-alpha-stimulated VEGF secretion.

CONCLUSIONS

These results suggest that IL-17 is an in vitro stimulator of angiogenic factor release, both by its own action and by cooperating with TNF-alpha.

<em>Growth</em> <em>factors</em> and their receptors play important roles in cell proliferation, migration, tissue injury repair and ulcer healing. In gastric mucosa, transforming <em>growth</em> <em>factor</em> alpha (TGF-alpha) and epidermal <em>growth</em> <em>factor</em> (EGF) by activating their common receptor, control cell proliferation. TGF-alpha predominantly plays this role under normal conditions and after acute injury, while EGF exerts its actions mainly during healing of chronic ulcers. During regeneration of injured gastric mucosa, these <em>growth</em> <em>factors</em> serve predominantly to restore the epithelial component. Other <em>growth</em> <em>factors</em>, basic fibroblast <em>growth</em> <em>factor</em> (bFGF) and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) serve to promote restoration of the connective tissue and microvessels (angiogenesis) in injured mucosa. During healing of chronic ulcers, a new epithelial lineage secreting EGF and other <em>growth</em> peptides develops and the majority of cells lining the ulcer margin overexpress the EGF receptor. Activation of the EGF receptor induces dramatic increases in MAP (Erk -1 and -<em>2</em>) kinase activity and phosphorylation levels. Inhibition of this signaling pathway dramatically delays ulcer healing. Granulation connective tissue, which grows under the stimulation of bFGF and VEGF is the major source for regeneration of connective tissue lamina propria and microvessels within the ulcer scar. Other <em>growth</em> <em>factors</em> such as insulin - like <em>growth</em> <em>factor</em>, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, hepatocyte <em>growth</em> <em>factor</em> and trefoil peptides have been implicated in gastrointestinal (gastric ulcers, colitis) regeneration following injury. This paper is intended to provide an overview of the role of <em>growth</em> <em>factors</em> in gastrointestinal mucosal regeneration.

The corneal epithelial basement membrane (BM) is positioned between basal epithelial cells and the stroma. This highly specialized extracellular matrix functions not only to anchor epithelial cells to the stroma and provide scaffolding during embryonic development but also during migration, differentiation, and maintenance of the differentiated epithelial phenotype. Basement membranes are composed of a diverse assemblage of extracellular molecules, some of which are likely specific to the tissue where they function; but in general they are composed of four primary components--collagens, laminins, heparan sulfate proteoglycans, and nidogens--in addition to other components such as thrombospondin-1, matrilin-<em>2</em>, and matrilin-4 and even fibronectin in some BM. Many studies have focused on characterizing BM due to their potential roles in normal tissue function and disease, and these structures have been well characterized in many tissues. Comparatively few studies, however, have focused on the function of the epithelial BM in corneal physiology. Since the normal corneal stroma is avascular and has relatively low keratocyte density, it is expected that the corneal BM would be different from the BM in other tissues. One function that appears critical in homeostasis and wound healing is the barrier function to penetration of cytokines from the epithelium to stroma (such as transforming <em>growth</em> <em>factor</em> β-1), and possibly from stroma to epithelium (such as <em>keratinocyte</em> <em>growth</em> <em>factor</em>). The corneal epithelial BM is also involved in many inherited and acquired corneal diseases. This review examines this structure in detail and discusses the importance of corneal epithelial BM in homeostasis, wound healing, and disease.

Angiogenesis is a prominent feature of a number of inflammatory human diseases, including rheumatoid arthritis, psoriasis, and cutaneous delayed-type hypersensitivity (DTH) reactions. Up-regulation of placental <em>growth</em> <em>factor</em> (PlGF), a member of the vascular endothelial <em>growth</em> <em>factor</em> (VEGF) family, has been found in several conditions associated with pathologic angiogenesis; however, its distinct role in the control of angiogenesis has remained unclear. To directly investigate the biologic function of PlGF in cutaneous inflammation and angiogenesis, DTH reactions were investigated in the ear skin of wild-type mice, of PlGF-deficient mice, and of transgenic mice with targeted overexpression of human PlGF-<em>2</em> in epidermal <em>keratinocytes</em>, driven by a keratin 14 promoter expression construct. Chronic transgenic delivery of PlGF-<em>2</em> to murine epidermis resulted in a significantly increased inflammatory response, associated with more pronounced vascular enlargement, edema, and inflammatory cell infiltration than seen in wild-type mice. Conversely, PlGF deficiency resulted in a diminished and abbreviated inflammatory response, together with a reduction of inflammatory angiogenesis and edema formation. VEGF expression was up-regulated at a comparable level in the inflamed skin of all genotypes. These findings reveal that placental <em>growth</em> <em>factor</em> plays a critical role in the control of cutaneous inflammation, and they suggest inhibition of PlGF bioactivity as a potential new approach for anti-inflammatory therapy.

Mouse skin carcinomas arise from a small subpopulation of benign papillomas with an increased risk of malignant conversion. These papillomas arise with limited stimulation by tumor promoters, appear rapidly, and do not regress, suggesting that they differ in <em>growth</em> properties from the majority of benign tumors. The transforming <em>growth</em> <em>factor</em> beta (TGF-beta) proteins are expressed in the epidermis and are <em>growth</em> inhibitors for mouse <em>keratinocytes</em> in vitro; altered TGF-beta expression could influence the <em>growth</em> properties of high-risk papillomas. Normal epidermis, tumor promoter-treated epidermis, and skin papillomas at low risk for malignant conversion express TGF-beta 1 in the basal cell compartment and TGF-beta <em>2</em> in the suprabasal strata. In low-risk tumors, 90% of the proliferating cells are confined to the basal compartment. In contrast, the majority of high-risk papillomas are devoid of both TGF-beta 1 and TGF-beta <em>2</em> as soon as they arise; these tumors have up to 40% of the proliferating cells in the suprabasal layers. Squamous cell carcinomas are also devoid of TGF-beta, suggesting that they arise from the TGF-beta-deficient high-risk papillomas. In some high-risk papillomas, TGF-beta 1 loss can occur first and correlates with basal cell hyperproliferation, while TGF-beta <em>2</em> loss correlates with suprabasal hyperproliferation. Similarly, TGF-beta 1-null transgenic mice, which express wild-type levels of TGF-beta <em>2</em> in epidermis but no TGF-beta 1 in the basal layer, have a hyperproliferative basal cell layer without suprabasal proliferation. In tumors, loss of TGF-beta is controlled at the posttranscriptional level and is associated with expression of keratin 13, a documented marker of malignant progression. These results show that TGF-beta expression and function are compartmentalized in epidermis and epidermal tumors and that loss of TGF-beta is an early, biologically relevant risk <em>factor</em> for malignant progression.

One of the more intriguing aspects of transforming <em>growth</em> <em>factor</em> beta 1 (TGF beta 1) is its ability to function as both a mitogenic <em>factor</em> for certain mesenchymal cells and a potent <em>growth</em> inhibitor of lymphoid, endothelial, and epithelial cells. Data are presented indicating that c-myc may play a pivotal role in both the mitogenic and antiproliferative actions of TGF beta 1. In agreement with previous studies using C3H/10T1/<em>2</em> fibroblasts constitutively expressing an exogenous c-myc cDNA, we show that AKR-<em>2</em>B fibroblasts expressing a chimeric estrogen-inducible form of c-myc (mycER) are able to form colonies in soft agar in the presence of TGF beta 1 only when c-myc is activated by hormone. Whereas these findings support a synergistic role for c-myc in mitogenic responses to TGF beta 1, we also find that c-myc can antagonize the <em>growth</em>-inhibitory response to TGF beta 1. Mouse <em>keratinocytes</em> (BALB/MK), which are normally <em>growth</em>-arrested by TGF beta 1, are rendered insensitive to the <em>growth</em>-inhibitory effects of TGF beta 1 upon mycER activation. This ability of mycER activation to block TGF beta 1-induced <em>growth</em> arrest was found to occur only when the fusion protein was induced with hormone in the early part of G1. Addition of estradiol late in G1 had no suppressive effect on TGF beta 1-induced <em>growth</em> inhibition.

Chronic exposure to ultraviolet (UV) irradiation induces skin cancer, in part, through epigenetic mechanisms that result in the deregulation of cell proliferation. UV irradiation also rapidly activates the epidermal <em>growth</em> <em>factor</em> receptor (EGFR). Since EGFR activation is strongly mitogenic in many cell types including <em>keratinocytes</em> of the skin, we hypothesized that UV-induced cutaneous proliferation results from EGFR activation. The role of EGFR activation in the response of the skin to UV was determined using Egfr-null and Egfr-wild-type skin grafted onto athymic nude mouse hosts, because Egfr-null mice survive only a few days after birth. EGFR was rapidly activated in mouse epidermis following exposure to UV, as detected by the phosphorylation of EGFR on tyrosine residues 99<em>2</em>, 1045, 1068 and 1173. UV induced epidermal hyperplasia in Egfr-wild-type skin between 48 and 7<em>2</em> h post-UV. However, no epidermal hyperplasia occurred in Egfr-null skin. Baseline cell proliferation was similar in skin grafts of both genotypes. However, UV exposure increased cell proliferation, as measured by Ki67 immunohistochemistry and proliferating cell nuclear antigen immunoblotting, maximally at 48 h to a level more than three times higher in wild-type compared with Egfr-null skin. Apoptotic cell death, as measured by terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) analysis, was also increased in UV-exposed Egfr-null skin when compared with wild-type 1-<em>2</em> days post-UV. These changes in cellular homeostasis after UV were accompanied by increased cyclin D expression in wild-type but not Egfr-null skin and increased expression of p53 and the cyclin-dependent kinase (CDK) inhibitor p<em>2</em>1waf1 in Egfr-null skin when compared with wild-type. Collectively, these results demonstrate that the UV-induced activation of EGFR augments <em>keratinocyte</em> proliferation and suppresses apoptosis, leading to epidermal hyperplasia, associated with increased G1 cyclin expression and suppression of CDK inhibitor expression.