The prostaglandin E(<em>2</em>) (PGE(<em>2</em>)) G protein-coupled receptor (GPCR), EP<em>2</em>, plays important roles in mouse skin tumor development (Chun, K. S., Lao, H. C., Trempus, C. S., Okada, M., and Langenbach, R. (<em>2</em>009) Carcinogenesis 30, 16<em>2</em>0-16<em>2</em>7). Because <em>keratinocyte</em> proliferation is essential for skin tumor development, EP<em>2</em>-mediated signaling pathways that contribute to <em>keratinocyte</em> proliferation were investigated. A single topical application of the EP<em>2</em> agonist, butaprost, dose-dependently increased <em>keratinocyte</em> replication via activation of epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and PKA signaling. Because GPCR-mediated activation of EGFR can involve the formation of a GPCR-β-arrestin-Src signaling complex, the possibility of a β-arrestin1-Src complex contributing to EP<em>2</em>-mediated signaling in <em>keratinocytes</em> was investigated. Butaprost induced β-arrestin1-Src complex formation and increased both Src and EGFR activation. A role for β-arrestin1 in EP<em>2</em>-mediated Src and EGFR activation was demonstrated by the observation that β-arrestin1 deficiency significantly reduced Src and EGFR activation. In agreement with a β-arrestin1-Src complex contributing to EGFR activation, Src and EGFR inhibition (PP<em>2</em> and AG1478, respectively) indicated that Src was upstream of EGFR. Butaprost also induced the activation of Akt, ERK1/<em>2</em>, and STAT3, and both β-arrestin1 deficiency and EGFR inhibition (AG1478 or gefitinib) decreased their activation. In addition to β-arrestin1-dependent EGFR activation, butaprost increased PKA activation, as measured by phospho-GSK3β (p-GSK3β) and p-cAMP-response element-binding protein formation. PKA inhibition (H89 or R(P)-adenosine-3',5'-cyclic monophosphorothioate (R(P)-cAMPS)) decreased butaprost-induced cAMP-response element-binding protein and ERK activation but did not affect EGFR activation, whereas β-arrestin1 deficiency decreased EGFR activation but did not affect butaprost-induced PKA activation, thus indicating that they were independent EP<em>2</em>-mediated pathways. Therefore, the results indicate that EP<em>2</em> contributed to mouse <em>keratinocyte</em> proliferation by G protein-independent, β-arrestin1-dependent activation of EGFR and G protein-dependent activation of PKA.

We examined in situ expression of putative hyaluronan synthase genes, Has1 and Has<em>2</em>, and effects of transforming <em>growth</em> <em>factor</em>-beta on their expression. In situ mRNA hybridization showed that mouse skin expressed both Has1 and Has<em>2</em> mRNA in dermis and epidermis. In dermis, the number of cells expressing the Has1 mRNA was less than that of the Has<em>2</em> mRNA, and in epidermis, some strong signals from both mRNA were seen in stratum granulosum. Northern blot analysis showed that cultured human skin fibroblasts expressed Has1 mRNA of <em>2</em>.4 kb and Has<em>2</em> mRNA of 3.<em>2</em> and 4.8 kb, whereas human <em>keratinocytes</em> expressed Has1 mRNA of 4.8 but not <em>2</em>.4 kb and a trace of Has<em>2</em> mRNA. When the cultures were stimulated with transforming <em>growth</em> <em>factor</em>-beta, both Has1 and Has<em>2</em> mRNA were upregulated in fibroblasts, and only Has1 mRNA of <em>2</em>.4 but not 4.8 kb was induced in <em>keratinocytes</em>. The maximal amount of the upregulated Has1 mRNA in <em>keratinocytes</em> at <em>2</em> h after stimulation decreased time-dependently to the nonstimulated level at 18 h, although the stimulation for 18 h of fibroblasts was effective on the expression of both Has mRNA. Differences in expression pattern of Has and Has<em>2</em> mRNA in mouse skin and a higher response of fibroblasts to transforming <em>growth</em> <em>factor</em>-beta suggest that Has1 and Has<em>2</em> genes are regulated independently and synthesized hyaluronan may have a different function in epidermis and dermis.

BACKGROUND

Keratinocyte growth factor (KGF; palifermin) is a growth factor with a high degree of specificity for epithelial cells. KGF is an important effector of epithelial growth and tissue homeostasis in various organs including the pancreas. Here we investigated the intracellular signaling pathways involved in the mediation of pancreatic ductal cell proliferation and differentiation induced by exogenous KGF during beta-cell regeneration in diabetic rat.

RESULTS

In vitro and in vivo duct cell proliferation was measured by BrdU incorporation assay. The implication of MAPK-ERK1/2 in the mediation of KGF-induced cell proliferation was determined by inactivation of this pathway, using the pharmacological inhibitor or antisense morpholino-oligonucleotides against MEK1. In vivo KGF-induced duct cell differentiation was assessed by the immunolocalization of PDX1 and Glut2 in ductal cells and the implication of PI3K/AKT in this process was investigated. We showed that KGF exerted a potent mitogenic effect on ductal cells. Both in vitro and in vivo, its effect on cell proliferation was mediated through the activation of ERK1/2 as evidenced by the abolition of duct cell proliferation in the context of MEK/ERK inactivation. In vivo, KGF treatment triggered ductal cell differentiation as revealed by the expression of PDX1 and Glut2 in a subpopulation of ductal cells via a PI3K-dependent mechanism.

CONCLUSIONS

Here we show that KGF promotes beta-cell regeneration by stimulating duct cell proliferation in vivo. Moreover, we demonstrated for the first time that KGF directly induces the expression of PDX1 in some ductal cells thus inducing beta-cell neogenesis. We further explored the molecular mechanisms involved in these processes and showed that the effects of KGF on duct cell proliferation are mediated by the MEK-ERK1/2 pathway, while the KGF-induced cell differentiation is mediated by the PI3K/AKT pathway. These findings might have important implications for the in vivo induction of duct-to-beta cell neogenesis in patients with beta-cell deficiency.

The effects of dermal fibroblasts on <em>keratinocyte</em> out<em>growth</em> on collagen substrata was studied using an in vitro <em>keratinocyte</em>-collagen gel composite model. Skin fibroblasts were seeded inside collagen gels, which remained attached to the cell culture plastic substratum. Fibroblasts incorporated in collagen gels were either kept viable throughout the study, or were lysed hypotonically with water at different time intervals (<em>2</em> hours and 5 days). Results show that very little <em>keratinocyte</em> out<em>growth</em> occurred on either plain collagen gels or gels that had previously contained viable fibroblasts for <em>2</em> hours. A 3- to 4-fold increase in <em>keratinocyte</em> out<em>growth</em> occurred on collagen gels that had previously contained viable fibroblasts for 5 days. A striking increase (<em>2</em>0-fold) in <em>keratinocyte</em> out<em>growth</em> was observed on collagen gels that contain viable fibroblasts. The effect of fibroblast diffusible <em>factors</em> on <em>keratinocyte</em> out<em>growth</em> was further studied with a co-culture system using Millicell inserts. It was found that the co-culture of fibroblasts with the composite enhanced <em>keratinocyte</em> out<em>growth</em> on collagen gels that had previously contained viable fibroblasts for 5 days. Among all, however, the <em>keratinocyte</em> out<em>growth</em> was far better on gels containing viable fibroblasts. Addition of <em>keratinocyte</em> <em>growth</em> <em>factor</em> or its neutralizing antibody did not affect <em>keratinocyte</em> out<em>growth</em>. These results suggest that dermal fibroblasts can activate <em>keratinocyte</em> out<em>growth</em> on collagen matrices through some diffusible <em>factors</em> other than <em>keratinocyte</em> <em>growth</em> <em>factor</em>, and epithelial-mesenchymal interactions exert some special effects on <em>keratinocyte</em> out<em>growth</em> on collagen gels.

Indole-3-carbinol (I3C) and its dimer 3,3'-diindolylmethane (DIM), obtained from dietary consumption of cruciferous vegetables, have multiple biochemical activities. Both compounds have been effective clinically in treating precancerous lesions of the cervix and laryngeal papillomas, pathologies with a human papillomavirus (HPV) component. Using cDNA microarrays, we examined early changes in gene expression after treatment with 100 micro mol/L DIM in C33A and CaSki cervical cancer cells and in an immortalized human epithelial cell line (HaCat), as well as in normal human foreskin <em>keratinocytes</em> (HFK). Multiple analyses were done after treating C33A cells for 6 h; other analyses included 4- and 1<em>2</em>-h treatments of C33A and 6-h treatments of CaSki, HaCat and HFK cells. DIM consistently altered the expression of >100 genes at least twofold. Many of the stimulated genes encode transcription <em>factors</em> and proteins involved in signaling, stress response and <em>growth</em>. Results were comparable between transformed cells with and without integrated HPV sequences, and many of the same genes were induced in these cancer-derived cells and in noncancer cells. Eight genes encoding bZip proteins were among the most consistently and robustly induced, including the stress-associated immediate early gene GADD153 (>50 fold in C33A) and nuclear <em>factor</em>-interleukin 6 (NF-IL6), also known as c/EBPbeta, (>5 fold in C33A), which has been shown to reduce expression of HPV oncogenes. Induction of GADD153, NF-IL6 and ATF3 was confirmed by Western analysis. In functional analyses, DIM not only suppressed transcription of a luciferase gene driven by the HPV11 upstream regulatory region (URR) in C33A, CaSki, HaCat and HFK cells from>><em>2</em>-fold to 37-fold depending on the type of cells, but also reduced endogenous transcription of HPV16 oncogenes to undetectable levels in CaSki cells as determined by an RNase protection assay. Ectopic expression of GADD153 or NF-IL6 suppressed transcription in a dose-dependent manner driven by the HPV11 URR in C33A, CaSki, HaCat and HFK cells. These results identify unexpected ways in which dietary I3C and DIM invoke cellular responses and are consistent with a potential antiviral effect of DIM on <em>keratinocytes</em>, but they do not explain the differential sensitivity of transformed <em>keratinocytes</em> to apoptosis by DIM.

Glucocorticoids exert a deleterious effect on the wound healing process, which has been suggested to result from the anti-inflammatory action of these steroids. In addition, recent studies have demonstrated that glucocorticoids regulate the expression of various genes at the wound site which are likely to encode key players in the wound repair process. Using a murine full-thickness excisional wound healing model, we analyzed the effect of dexamethasone on the expression of various cytokines, <em>growth</em> <em>factors</em>, enzymes, and extracellular matrix molecules in normal and wounded skin. We demonstrate that the proinflammatory cytokines interleukin-1 alpha and -beta, tumor necrosis <em>factor</em> alpha, <em>keratinocyte</em> <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factors</em> beta 1, beta <em>2</em>, and beta 3 and their receptors, platelet-derived <em>growth</em> <em>factors</em> and their receptors, tenascin-C, stromelysin-<em>2</em>, macrophage metalloelastase, and enzymes involved in the generation of nitric oxide are targets of glucocorticoid action in wounded skin. These results indicate that anti-inflammatory steroids inhibit wound repair at least in part by influencing the expression of these key regulatory molecules.

BALB-/MK-<em>2</em> mouse epidermal <em>keratinocytes</em> required epidermal <em>growth</em> <em>factor</em> for proliferation and terminally differentiated in response to high Ca<em>2</em>+ concentration. Infection with retroviruses containing transforming genes of the src and ras oncogene families led to rapid loss of epidermal <em>growth</em> <em>factor</em> dependence, in some cases, accompanied by alterations in cellular morphology. The virus-altered cells continued to proliferate in the presence of high levels of extracellular calcium but exhibited alterations in normal <em>keratinocyte</em> terminal differentiation that appear to be specific to the particular oncogene. These alterations bore similarities to abnormalities in differentiation observed in naturally occurring squamous epithelial malignancies.

Although originally conceived as a basis for malignant cell <em>growth</em>, autocrine signaling networks are currently known to be activated during tissue repair and with in vitro cultivation. In human epidermal <em>keratinocytes</em>, activation of the epidermal <em>growth</em> <em>factor</em> receptor by cognate ligands mediates the majority of the autonomous replicative capacity of these cells and is necessary to inhibit differentiation and apoptosis. The importance of heparin-binding <em>growth</em> <em>factors</em> in activation of this receptor was first suggested by the strong anti-proliferative effects of soluble heparin-like molecules on <em>keratinocyte</em> <em>growth</em>. This and related evidence led to the identification of amphiregulin as a major autocrine <em>factor</em> for <em>keratinocytes</em>. The binding of amphiregulin and its homolog, heparin-binding epidermal <em>growth</em> <em>factor</em>-like <em>growth</em> <em>factor</em>, to the receptor is potentially amplified by autoinduction and cross-signaling through epidermal <em>growth</em> <em>factor</em>-related polypeptides and by transmodulation of other ErbB-family receptors (HER-<em>2</em>, -3, -4) in cells expressing these receptors. Heparan sulfate proteoglycans and the tetraspanin family of membrane-associated proteins appear to act as co<em>factors</em> in amphiregulin-driven mitogenesis mediated by the epidermal <em>growth</em> <em>factor</em> receptor, but amphiregulin's immunolocalization to <em>keratinocyte</em> nuclei and to filopodia may indicate other potentially novel effects. Following from the observation that amphiregulin is overexpressed in lesional psoriatic epidermis, the importance of amphiregulin in hyperproliferative skin diseases has been further supported by recent studies of the targeted expression of a transgene encoding keratin 14 promoter-driven human amphiregulin to the basal epidermis of mice. Founder transgenic mice displayed a morphologic and microscopic cutaneous phenotype that shares characteristics with psoriasis. Pharmacologic regulation of amphiregulin's expression and receptor signaling may eventually prove to be an effective strategy in the treatment of hyperproliferative skin diseases.

Human <em>keratinocytes</em> (HK) migration plays a critical role in the re-epithelialization of acute skin wounds. Although extracellular matrices (ECM) and <em>growth</em> <em>factors</em> (GF) are the two major pro-motility signals, their functional relationship remains unclear. We investigated how ECM and GF regulate HK motility under defined conditions: (1) in the absence of GF and ECM and (<em>2</em>) with or without GF with cells apposed to a known pro-motility ECM. Our results show that HK migrate on selected ECM even in the total absence of GF. This suggests that certain ECM alone are able to "initiate" HK migration. Unlike ECM, however, GF alone cannot initiate HK migration. HK cannot properly migrate when plated in the presence of GF, regardless of the concentration, without an ECM substratum. The role of GF, instead, is to augment ECM-initiated motility and provide directionality. To gain insights into the mechanism of action by ECM and GF, we compared, side-by-side, the roles of three major mitogen-activated protein kinase cascades, extracellular-signal-regulated kinase (ERK)1/<em>2</em>, p38, and c-Jun N-terminal kinase (JNK). Our data show that ERK1/<em>2</em> is involved in mediating collagen's initiation signal and GF's augmentation signal. p38 is specific for GF's augmentation signal. JNK is uninvolved in HK motility. Constitutively activated p38 and ERK1/<em>2</em> alone could not initiate HK migration. Co-expression of both constitutively activated p38 and ERK1/<em>2</em>, however, could partially mimic the pro-motility effects of collagen and GF. This study reveals for the first time the specific functions of ECM and GF in cell motility.

Proteolytic processing of the Alzheimer amyloid precursor protein (APP) results in the generation of at least two distinct classes of biologically relevant peptides: (1) the amyloid beta peptides which are believed to be involved in the pathogenesis of Alzheimer's disease and (<em>2</em>) the soluble N-terminal ectodomain (sAPP) which exhibits a protective but as yet ill-defined effect on neurons and epithelial cells. In this report we present an overview on the functions of sAPP as an epithelial <em>growth</em> <em>factor</em>. This function involves specific binding of sAPP to membrane rafts and results in signal transduction and various physiological effects in epithelial cells as different as <em>keratinocytes</em> and thyrocytes. At nanomolar concentrations sAPP induces a two to fourfold increase in the rate of cell proliferation and cell migration. Specific inhibition of APP expression by antisense techniques results in decreased sAPP release and in reduced proliferative and motogenic activities. Proliferation and migration are known to be part of complex processes such as wound healing which, therefore, might be facilitated by the <em>growth</em> <em>factor</em> function of sAPP.

OBJECTIVE

The purpose of this study was to use primary cultured rat alveolar epithelial cell monolayers to examine the potential of using transferrin receptor (TfR)-mediated transcytosis for noninvasive systemic protein drug delivery via the pulmonary route.

METHODS

Freshly isolated rat type II pneumocytes were plated onto tissue culture-treated polycarbonate 1<em>2</em>-mm Transwells. AEC monolayers >> or = <em>2</em>500 omega(cm<em>2</em>)) were treated with <em>keratinocyte</em> <em>growth</em> <em>factor</em> (10 ng/mL) for maintenance of type II cell-like characteristics. Filgrastim (GCSF)-Tf conjugates were prepared using the linkers SPDP and DPDPB. TfR-specific binding and uptake were determined using 1<em>2</em>5I-Tf and 59Fe-Tf treatment, respectively. Apical-to-basolateral (A-to-B) transferrin receptor (TfR)-mediated transcytosis was determined by dosing the apical compartment with 1.5 microg/mL of 1<em>2</em>5I-Tf or 1<em>2</em>5I-GCSF-Tf. Nonspecific TfR-independent transport of 1<em>2</em>5I-Tf and 1<em>2</em>5I-GCSF-Tf was determined in parallel by including 150 microg/mL of nonradiolabeled Tf. Basolateral samples (500 microL) were taken at <em>2</em>, 4, and 6 h post-dosing, subjected to 15% trichloroacetic acid precipitation, and assayed in a Packard gamma counter. TfR-specific transport was determined as the difference between total and nonspecifc transport. The effects of brefeldin-A (BFA) on TfR distribution and (A-to-B) transport of 1<em>2</em>5I-Tf, 1<em>2</em>5I-GCSF and 1<em>2</em>5I-GCSF-Tf was studied by including the agent in the apical fluid at 1 microg/mL.

RESULTS

BFA treatment resulted in a small significant reduction in TfR at the basolateral surface of type II cell-like monolayers, while it had no effect on TfR distribution in type I cell-like monolayers. In contrast, BFA treatment significantly altered the endocytosis of TfR, reducing the basolateral uptake of 59Fe-Tf while greatly increasing the apical uptake of 59Fe-Tf. BFA treatment, however, did not affect the TfR-specific uptake of 59Fe-Tf in type I cell-like monolayers. TfR-specific apical-to-basolateral transcytosis of 1<em>2</em>5I-Tf and 1<em>2</em>6I-GCSF-Tf conjugates was significantly enhanced in the presence of BFA in type II cell-like monolayers, whereas it had no effect on apical-to-basolateral transport of 1<em>2</em>5I-GCSF. BFA-enhanced transport of 1<em>2</em>5I-GCSF-Tf was approximately 3-fold higher than that of 1<em>2</em>5I-GCSF in the presence or absence of BFA. Moreover, 1<em>2</em>5I-GCSF transport in the presence of BFA was not significantly different from non-specific 1<em>2</em>5I-GCSF-Tf transport. Chromatographic analyses and bio-assays revealed that GCSF-Tf was not degraded during transport via TfR-specific processes, and that GCSF retained biologic activity when liberated from the conjugate via dithiothreitol reduction.

CONCLUSIONS

This study suggests the possibility of using TfR-mediated transcytosis for systemic delivery of therapeutic proteins via the alveolar epithelium.

<em>Keratinocyte</em> terminal differentiation is the process that ultimately forms the epidermal barrier that is essential for mammalian survival. This process is controlled, in part, by signal transduction and gene expression mechanisms, and the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Using microarray analysis of a confluent cell density-induced model of <em>keratinocyte</em> differentiation, we identified <em>2</em>,676 genes that are regulated by epidermal <em>growth</em> <em>factor</em> (EGF), a ligand of the EGFR. We further discovered, and separately confirmed by functional assays, that EGFR activation abrogates all of the known essential processes of <em>keratinocyte</em> differentiation by 1) decreasing the expression of lipid matrix biosynthetic enzymes, <em>2</em>) regulating numerous genes forming the cornified envelope, and 3) suppressing the expression of tight junction proteins. In organotypic cultures of skin, EGF acted to impair epidermal barrier integrity, as shown by increased transepidermal water loss. As defective epidermal differentiation and disruption of barrier function are primary features of many human skin diseases, we used bioinformatic analyses to identify genes that are known to be associated with skin diseases. Compared with non-EGF-regulated genes, EGF-regulated genes were significantly enriched for skin disease genes. These results provide a systems-level understanding of the actions of EGFR signaling to inhibit <em>keratinocyte</em> differentiation, providing new insight into the role of EGFR imbalance in skin pathogenesis.

Epidermal <em>growth</em> <em>factor</em> receptor (EGFR) ligands are fundamental regulators of epithelial <em>growth</em>, differentiation, and neoplastic transformation. In addition to being potent mitogens for murine epidermal <em>keratinocytes</em> in vitro, transforming <em>growth</em> <em>factor</em> alpha (TGF alpha) and EGF elicit distinctive changes in keratin expression: Ca(<em>2</em>+)-mediated induction of the differentiation-specific keratins K1 and K10 is blocked, while simple epithelial keratins K8 and K18 are expressed aberrantly (C. Cheng et al., Cell <em>Growth</em>, & Differ., 4: 317-3<em>2</em>7, 1993). We have evaluated several additional <em>growth</em> <em>factors</em> to determine the specificity of this response for EGFR ligands. TGF alpha, <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF), and acidic fibroblast <em>growth</em> <em>factor</em> (aFGF), but not basic fibroblast <em>growth</em> <em>factor</em> (bFGF) or insulin-like <em>growth</em> <em>factor</em> type I, block Ca(<em>2</em>+)-mediated expression of K1 while inducing K8. Since KGF and aFGF (but not bFGF) are ligands for the KGF receptor (KGFR), we explored the possibility that the TGF alpha/EGFR pathway is an intermediary in signaling through the KGFR. TGF alpha mRNA was increased in cells treated with KGF, aFGF, or TGF alpha but not bFGF or insulin-like <em>growth</em> <em>factor</em> type I. Similar changes were detected at the protein level; TGF alpha in conditioned medium (CM) from control, KGF-, TGF alpha-, and aFGF-treated cultures was 54 (+/- 8, SEM), 365 (+/- 50), 146 (+/- <em>2</em>0), and 1<em>2</em>0 (+/- 50) pg/ml, respectively. KGF and TGF alpha also increased expression of cell-associated TGF alpha measured in <em>keratinocyte</em> lysates. KGF increased TGF alpha secretion and mRNA levels in human as well as mouse <em>keratinocytes</em>. CM from KGF-treated cultures stimulated cell <em>growth</em> when added to cultures of normal <em>keratinocytes</em>. Preincubation with neutralizing antibodies to both TGF alpha and KGF, but not KGF antibody alone, blocked cell <em>growth</em> in cultures treated with KGF CM, suggesting that the predominant <em>keratinocyte</em> mitogen in KGF CM is TGF alpha. In support of this hypothesis, treatment of <em>keratinocytes</em> for 5 min with either KGF CM or purified TGF alpha resulted in EGFR autophosphorylation. Furthermore, after approximately <em>2</em>4 h, KGF as well as TGF alpha induced EGFR down-regulation based on Western blot analysis and 1<em>2</em>5I-EGF binding. Induction of TGF alpha in KGF-treated <em>keratinocytes</em>, coupled to activation and down-modulation of the EGFR, suggests that TGF alpha may be a proximal effector of KGF action for at least certain aspects of epidermal <em>growth</em> and differentiation.

Hepatocyte <em>growth</em> <em>factor</em> (HGF/SF)-induced expression of vascular endothelial <em>growth</em> <em>factor</em> (VEGF/VPF) has been implicated in paracrine amplification of angiogenesis, contributing to angiogenic responses during inflammation, wound healing, collateral formation and tumor <em>growth</em>. We have shown previously that HGF/SF-mediated VEGF/VPF expression by <em>keratinocytes</em> is primarily dependent on transcriptional activation, and we mapped the HGF/SF-responsive element to a GC-rich region between bp -88 and -65. Sp1-like <em>factors</em> bind to this element constitutively; however the VEGF/VPF promoter is transactivated by HGF/SF in the absence of induced binding activity. In experimental approaches to clarify molecular mechanisms of Sp1-dependent VEGF/VPF gene transcription, neither HGF/SF-dependent changes in nuclear expression nor in relative DNA binding activity of Sp family members to the indicated element were observed. Thus, HGF/SF was hypothesized to induce VEGF/VPF gene transcription via increased transactivation activity of Sp1 owing to biochemical modification. In immunoprecipitation studies, HGF/SF was found to increase the amount of serine-phosphorylated Sp1, revealing a likely mechanism of HGF/SF-induced VEGF/VPF expression, as phosphorylation may enhance the transcriptional activity of Sp1. The contribution of different signaling molecules to HGF/SF-induced VEGF/VPF transcription was demonstrated by the use of chemical inhibition, of expression of kinase-deficient signaling proteins, and by the use of antisense oligonucleotides. Herein, we provide evidence that PI 3-kinase, MEK1/<em>2</em> and PKC-zeta play a significant role in HGF/SF-induced VEGF/VPF promoter activation. Together, our results elucidate a critical pathway of paracrine amplification of angiogenesis, suggesting that HGF/SF-induced Sp1 phosphorylation may activate VEGF/VPF promoter activity that requires the contribution of distinct signaling molecules.

Laminin 5, an anchoring filament protein previously known as nicein/kalinin/epiligrin, consists of three polypeptide chains, alpha 3, beta 3, and gamma <em>2</em>, encoded by the genes LAMA3, LAMB3, and LAMC<em>2</em>, respectively. The expression of laminin 5 was detected by Northern hybridization with specific cDNA probes in various epidermal <em>keratinocyte</em> cultures, whereas no expression of any of the three genes could be detected in foreskin fibroblast cultures. Transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) enhanced LAMA3, LAMB3, and LAMC<em>2</em> gene expression in human epidermal <em>keratinocytes</em>, as well as in HaCaT and Balb/K cells in culture, although the extent of enhancement was greater for LAMA3 and LAMC<em>2</em> genes than for LAMB3. Interestingly, tumor necrosis <em>factor</em>-alpha, (TNF-alpha) alone did not alter the expression of LAMB3 and LAMC<em>2</em> genes in human epidermal <em>keratinocytes</em>, whereas it inhibited the expression of LAMA3. These results suggest that the expression of the three genes encoding the laminin 5 subunits is not coordinately regulated by the cytokines tested.

Upregulation of acidic and basic fibroblast <em>growth</em> <em>factors</em> (FGF-1 and -<em>2</em>), and their cognate receptors FGFR-1 and -<em>2</em>, has been demonstrated in a variety of epithelial malignancies. However, the patterns of FGF/FGFR expression at specific stages of epithelial carcinogenesis have not been extensively characterized. In this report, the levels of FGF-1, FGF-<em>2</em>, FGF-7 mRNA and their receptors FGFR-1 and FGFR-<em>2</em>, were investigated during epidermal carcinogenesis in transgenic mice expressing the early region of the 'high risk' papillomavirus type 16 (HPV16) under control of the human keratin-14 enhancer/promoter (K14-HPV16 transgenic mice). FGF-1 was first upregulated in dysplasias, while FGF-<em>2</em> was constitutively expressed in non-transgenic, neoplastic, and malignant <em>keratinocytes</em> throughout carcinogenesis. Expression of FGF-7 was undetectable in non-transgenic epidermis, and remained at threshold levels at all stages of progression. In well differentiated squamous cancers, FGFR-1 was upregulated and co-localized with angiogenic capillaries in the dermis underlying dysplastic lesions and within papillary fronds of invasive cancers. In contrast, FGFR-1 was upregulated specifically within the malignant squamous cells of moderate-poorly differentiated squamous cancers. The expression of FGFR-<em>2</em> was essentially constitutive in both non-transgenic and neoplastic epidermis. Collectively the data suggest that the FGF/FGFR signaling pathways may potentially contribute to several facets of multi-stage epithelial carcinogenesis, including auto- or paracrine <em>growth</em> stimulation, upregulation of angiogenesis, and stromal remodeling.

To investigate whether <em>growth</em> <em>factors</em> derived from T cells in psoriatic lesions are able to stimulate <em>keratinocyte</em> <em>growth</em>, T-cell lines were initiated from lesional psoriasis skin and cloned by limiting dilution. Eight clones with good proliferative capacity out of 40 clones from one patient were stimulated. After <em>2</em>4 h, the conditioned medium was harvested and the <em>growth</em> modulatory effect of the conditioned medium on <em>keratinocytes</em> was assessed. Seven of the eight T-cell clones stimulated <em>keratinocyte</em> <em>growth</em> to an extent ranging from <em>2</em><em>2</em>% +/- 19 to 64% +/- 9 (mean +/- SD of three experiments) of maximal inducible <em>keratinocyte</em> <em>growth</em>, and one T-cell clone had no effect (-5% +/- <em>2</em>) on <em>keratinocyte</em> <em>growth</em>. <em>Keratinocyte</em> <em>growth</em> was also induced by T-cell clones obtained from two other patients. Several cytokines were tested in this system to determine which T-cell <em>growth</em> <em>factor</em> may induce the <em>keratinocyte</em> <em>growth</em>. None of the cytokines interferon-g, transforming <em>growth</em> <em>factor</em>-beta, interleukin (IL)-<em>2</em>, IL-3, IL-4, IL-6, IL-8, or granulocyte-macrophage colony stimulating <em>factor</em> alone was found to possibly be responsible for the T-cell-induced <em>keratinocyte</em> <em>growth</em>. Thus the nature of the T-cell <em>keratinocyte</em> <em>growth</em>-promoting stimulus remains to be elucidated.

Endothelin (ET)-1 is a <em>2</em>1-amino acid peptide which has vasoconstrictor and <em>growth</em> regulatory activity. Recently, cultured <em>keratinocytes</em> have been reported to express ET-1 and its receptor when irradiated by ultraviolet (UV) B. In order to further understand the role of ET-1 in vivo during UVB-induced inflammation, we examined the localization, intensity and time course of the expression levels of ET-1 and its binding sites in UVB-exposed BALB/c mouse skin. Frozen and paraffin sections prepared from mouse skin 48 h after treatment with UVB irradiation (0.36 or 0.7<em>2</em> J/cm<em>2</em>) or after injection with tumor necrosis <em>factor</em> (TNF)-alpha (1.0 microgram) or interleukin (IL)-1 alpha (0.05 microgram) were incubated with monoclonal anti-ET-1 IgG and then visualized by peroxidase staining. In normal skin, faint ET-1 immunoreactivity was observed in the epidermis, pilosebaceous structures and blood vessels. Upon exposure to UVB irradiation or administration of TNF-alpha injection or IL-1 alpha injection, such immunoreactivity was found to be significantly enhanced. Subsequently, the frozen sections were incubated with 1<em>2</em>5I ET-1 for 30 min, and visualized by autoradiographic technique. In normal skin, ET-1 weakly bound to the skin, while UVB irradiation and TNF-alpha injection significantly enhanced ET-1 binding in the epidermis, pilosebaceous structures and blood vessels. Time course experiments (1, <em>2</em>, 4 and 7 days) indicated that ET-1 immunoreactivity and ET-1 binding peaked 1 or <em>2</em> days after UVB irradiation or TNF-alpha injection. These results suggest that the up-regulated expression of ET-1 and its binding sites in the epidermis and pilosebaceous structures may act as an autocrine/paracrine <em>factor</em> during UVB-induced inflammation.

This study examines the effect of transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1) on the expression of Type I and II transglutaminase in normal human epidermal <em>keratinocytes</em> (NHEK cells). Treatment of undifferentiated NHEK cells with 100 pM TGF-beta 1 caused a 10- to 15-fold increase in the activity of a soluble transglutaminase. Based on its cellular distribution and immunoreactivity this transglutaminase was identified as Type II (tissue) transglutaminase. TGF-beta 1 did not enhance the levels of the membrane-bound Type I (epidermal) transglutaminase activity which is induced during squamous cell differentiation and did not increase Type II transglutaminase activity in differentiated NHEK cells. Several SV40 large T antigen-immortalized NHEK cell lines also exhibited a dramatic increase in transglutaminase Type II activity after TGF-beta 1 treatment; however, TGF-beta 1 did not induce any significant change in transglutaminase activity in the carcinoma-derived cell lines SCC-13, SCC-15, and SQCC/Y1. Half-maximal stimulation of transglutaminase Type II activity in NHEK cells occurred at a dose of 15 pM TGF-beta 1. TGF-beta <em>2</em> was about equally effective. This enhancement in transglutaminase activity was related to an increase in the amount of transglutaminase Type II protein as indicated by immunoblot analysis. Northern blot analyses using a specific cDNA probe for Type II transglutaminase showed that exposure of NHEK cells to TGF-beta 1 caused a marked increase in the mRNA levels of this enzyme which could be observed as early as 4 h after the addition of TGF-beta 1. Maximal induction of transglutaminase Type II mRNA occurred between 18 and <em>2</em>4 h. The increase in Type II transglutaminase mRNA levels was blocked by the presence of cycloheximide, suggesting that this increase in mRNA by TGF-beta 1 is dependent on protein synthesis.

Exogenous glucagon-like peptide-<em>2</em> receptor (GLP-<em>2</em>R) activation elicits proliferative and cytoprotective responses in the gastrointestinal mucosa and ameliorates experimental small and large bowel gut injury. Nevertheless, the essential physiological role(s) of the endogenous GLP-<em>2</em>R remain poorly understood. We studied the importance of the GLP-<em>2</em>R for gut <em>growth</em>, epithelial cell lineage allocation, the response to mucosal injury, and host-bacterial interactions in Glp<em>2</em>r(-/-) and littermate control Glp<em>2</em>r(+/+) mice. Glp<em>2</em>r(-/-) mice exhibit normal somatic <em>growth</em> and preserved small and large bowel responses to IGF-I and <em>keratinocyte</em> <em>growth</em> <em>factor</em>. However, Glp<em>2</em>r(-/-) mice failed to up-regulate intestinal epithelial c-fos expression in response to acute GLP-<em>2</em> administration and do not exhibit changes in small bowel conductance or small or large bowel <em>growth</em> after administration of GLP-<em>2</em>R agonists. The crypt and villus compartment and the numbers and localization of Paneth, enteroendocrine, and goblet cells were comparable in Glp<em>2</em>r(+/+) vs. Glp<em>2</em>r(-/-) mice. Although the severity and extent of colonic mucosal injury in response to 3% oral dextran sulfate was similar across Glp<em>2</em>r genotypes, Glp<em>2</em>r(-/-) mice exhibited significantly increased morbidity and mortality and increased bacterial translocation after induction of enteritis with indomethacin and enhanced mucosal injury in response to irinotecan. Moreover, bacterial colonization of the small bowel was significantly increased, expression of Paneth cell antimicrobial gene products was reduced, and mucosal bactericidal activity was impaired in Glp<em>2</em>r(-/-) mice. Although the Glp<em>2</em>r is dispensable for gut development and the response to colonic injury, Glp<em>2</em>r(-/-) mice exhibit enhanced sensitivity to small bowel injury, and abnormal host-bacterial interactions in the small bowel.

Areca (betel) was recently proved a carcinogenic substance by the International Agency for Research on Cancer. However, the signaling impact of areca in oral <em>keratinocyte</em> is still obscure. Mitogen-activated protein kinase superfamilies, including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK) and p38, together with transcription <em>factor</em> NF-kappaB, are important signaling elements. We examined the activation of these signaling pathways in OECM-1 and SAS oral <em>keratinocytes</em>, treated with ripe areca nut extract (ANE). In both cells, a rapid increase in JNK1 activity at 0.5 hr was noted following treatment of ANE. ERK was profoundly activated during 0.5-<em>2</em> hr in OECM-1 cells. Contrasting p38 activity was noted in these <em>2</em> cells. In both cells, ANE also activated NF-kappaB pathway in a biphasic manner, particularly for SAS cells. NF-kappaB was activated by approximately <em>2</em>- to 4-fold at 0.5-1 hr and a plateau or slight decrease of activity existed between 1 and 6 hr. Later, another higher episode of NF-kappaB activity was raised. This was accompanied with the rapid degradation in cytosolic IkappaBalpha as well as an increase of nuclear NF-kappaB in both cells. ANE treatment did not activate epidermal <em>growth</em> <em>factor</em> receptor signaling system, but blockage of NF-kappaB activation rendered the suppression of ANE-modulated COX-<em>2</em> upregulation in OECM-1. This study identified that ANE affected interactive signaling systems in oral keratonocytes that could be the pathogenetic basis for areca.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) is expressed by uterine endometrial epithelial cells during the estrous cycle and during pregnancy in pigs, whereas KGF receptor is expressed in conceptus trophectoderm and endometrial epithelia. In particular, KGF expression in the endometrium is highest on day 1<em>2</em> of pregnancy. This corresponds to the period of maternal recognition of pregnancy in pigs, which is signaled by large amounts of estrogen secreted by conceptus trophectoderm acting on the endometrium. Our hypothesis is that estrogens of conceptus origin stimulate endometrial epithelial KGF expression, and, in turn, secreted KGF stimulates proliferation and differentiation of conceptus trophectoderm. To determine the <em>factors</em> affecting KGF expression in the uterus, endometrial explants from gilts on day 9 of the estrous cycle were cultured in the presence of 17beta-estradiol, catechol estrogens, or progesterone. 17beta-Estradiol stimulated the expression of KGF (P < 0.05), whereas catechol estrogens had no effect (P>> 0.05). Between days 9 and 15 of pregnancy, proliferating cell nuclear antigen was abundant in conceptuses, but was barely detectable in uterine endometrial epithelia. To determine the effects of KGF on conceptus trophectoderm, porcine trophectoderm (pTr) cells were treated with recombinant rat KGF (rKGF). rKGF increased the proliferation of pTr cells (P < 0.01) as measured by [(3)H]thymidine incorporation. rKGF elicited phosphorylation of KGF receptor and activated the mitogen-activated protein kinase (ERK1/<em>2</em>) cascade in pTr cells. pTr cell differentiation was affected by rKGF, because it increased expression of urokinase-type plasminogen activator, a marker for differentiation in pTr cells. Collectively, these results indicate that estrogen, the pregnancy recognition signal from the conceptus in pigs, increases uterine epithelial KGF expression, and, in turn, KGF stimulates the proliferation and differentiation of conceptus trophectoderm.

Recent studies have shown that adult skin incubated in low-Ca<em>2</em>+ (0.15 mM) medium rapidly degenerates but that normal architecture is maintained when the tissue is incubated in high-Ca<em>2</em>+ medium (1.4 mM Ca<em>2</em>+). To investigate whether the skin cell-produced <em>growth</em> <em>factors</em> insulin-like <em>growth</em> <em>factor</em>-1 (IGF-1) and epidermal <em>growth</em> <em>factor</em> (EGF) play a role in these events, <em>2</em>-mm skin punch biopsies were obtained and maintained for 8 to 10 days in a basal medium containing 0.15 mM Ca<em>2</em>+ with and without <em>growth</em> <em>factors</em>, or containing 1.4 mM Ca<em>2</em>+ with and without antibodies to the same <em>growth</em> <em>factors</em>. In parallel experiments, cultured human <em>keratinocytes</em> were incubated for <em>2</em> days in the same basal medium in the presence or absence of the same <em>growth</em> <em>factors</em> and antibodies. Consistent with previous reports, organ cultures incubated in the low-Ca<em>2</em>+ (0.15 mM) medium rapidly degenerated. Neither IGF-1 nor EGF prevented the complete degeneration of epidermis and dermis in these organ cultures. Interestingly, the addition of an anti-IGF-1 receptor (IGF-1R) antibody to the organ cultures maintained in high-Ca<em>2</em>+ medium induced changes reminiscent of those seen when the organ cultures were maintained in low-Ca<em>2</em>+ medium, i.e. tissue degeneration. In contrast, antibodies to EGF receptor, used for comparison, only produced focal areas of epidermal necrosis. In vitro, IGF-1 is a known mitogen for <em>keratinocytes</em>. In cultured human <em>keratinocytes</em>, anti-IGF-1R antibody partially inhibited the IGF-1-mediated stimulation of human <em>keratinocyte</em> proliferation without affecting normal spontaneous <em>growth</em>. Additionally, IGF-1R immunolocalized to basal <em>keratinocytes</em> in vivo, exhibited specific binding to IGF-1 in vitro. This indicated a critical role for IGF-1R in both organ cultures ex vivo and cultured cells in vitro. Messenger RNA encoding both IGF-1 and IGF-1R were readily detected by RT-PCR in organ cultures incubated in both low- and high-Ca<em>2</em>+ medium. There were no detectable differences in IGF-1 mRNA in organ cultures <em>growing</em> in the low- or high-Ca<em>2</em>+ medium, but lower levels of IGF-1R mRNA were observed in the organ cultures maintained in low-Ca<em>2</em>+ medium than in those in high Ca<em>2</em>+ medium. These findings are consistent with homeostatic changes in the tissue grown under different calcium concentrations. IGF-1 mRNA was detected in several skin cell populations in vitro, even though it was undetectable in cultured <em>keratinocytes</em>. Taken together these findings indicate that (1) the IGF-1/ IGF-1R loop is critically involved in maintenance of human skin organ cultures ex vivo, and (<em>2</em>) IGF-1, locally produced by skin cells other than <em>keratinocytes</em>, interacts with its receptor, predominantly expressed in basal <em>keratinocytes</em>, to maintain tissue homeostasis.

Recently we demonstrated that endothelins secreted from human <em>keratinocytes</em> act as intrinsic mitogens and melanogens for human melanocytes in UVB-induced melanosis. We show here that UVA-induced melanosis is associated with other <em>keratinocyte</em>-derived <em>growth</em> <em>factors</em>, secretion of which is specifically stimulated after exposure of human <em>keratinocytes</em> to UVA. Medium conditioned by UVA-exposed human <em>keratinocytes</em> elicited a significant increase in DNA synthesis by cultured human melanocytes in a UVA dose-dependent manner. Analysis of endothelin-1 and interleukin (IL)-1 alpha in the conditioned medium by ELISA, both of which are major <em>keratinocyte</em>-derived cytokines involved in UVB-associated melanocyte activation, revealed that UVA exposure did not cause human <em>keratinocytes</em> to stimulate the secretion of the two cytokines. In contrast, the levels of several other cytokines such as IL-6, IL-8 and granulocyte/macrophage colony-stimulating <em>factor</em> (GM-CSF) were significantly increased in the conditioned medium of human <em>keratinocytes</em> after exposure to UVA at a dose of 1.0 J/cm<em>2</em>. The gel chromatographic profile of UVA-exposed <em>keratinocyte</em>-conditioned medium demonstrated that there were two <em>factors</em> (P-1 and P-<em>2</em>) with molecular masses of approx. <em>2</em>0 and 1 kDa respectively that stimulate DNA synthesis in human melanocytes, and the larger species (P-1) also increased melanization as assessed by [14C]thiouracil incorporation. Quantitative analysis of cytokines in chromatographic fractions by ELISA revealed the P-1 fraction to be consistent with the molecular mass profile of GM-CSF. Furthermore the stimulatory effect of the P-1 fraction on DNA synthesis in human melanocytes was neutralized by antibodies to GM-CSF, but not to basic fibroblast <em>growth</em> <em>factor</em> or stem cell <em>factor</em>. Binding and proliferation assays with recombinant GM-CSF demonstrated that human melanocytes possess specific binding sites for GM-CSF(Kd <em>2</em>.11 nM; binding sites, <em>2</em>.5-3.5 x 10(4) per cell), and recombinant GM-CSF at concentrations of more than 10 nM significantly stimulated DNA synthesis and melanization. These findings suggest that GM-CSF secreted by <em>keratinocytes</em> plays an essential role in the maintenance of melanocyte proliferation and UVA-induced pigmentation in the epidermis.