<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and KGF receptor (KGFR) have been implicated in cancer <em>growth</em> as well as tissue development and repair. In this study, we examined whether KGF and KGFR have a role in human pancreatic ductal adenocarcinoma (PDAC). KGFR mRNA was expressed in eight pancreatic cancer cell lines, whereas the KGF mRNA was detected in seven of the cell lines and was absent in MIA PaCa-<em>2</em> cells. KGFR and KGF immunoreactivity were localized in the cancer cells in 41.5 and 34.0% of patients, respectively. There was a significant correlation between KGFR or KGF immunoreactivity and venous invasion and a significant correlation between the presence of both markers and venous invasion, vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A expression, and poor prognosis. Exogenous KGF increased VEGF-A expression and release in MIA PaCa-<em>2</em> cells, and PANC-1 cells stably transfected to overexpress KGF-exhibited increased VEGF-A expression. Moreover, short hairpin-KGFR transfection in MIA PaCa-<em>2</em> cells reduced the stimulatory effect of exogenous KGF on VEGF-A expression. Short hairpin-KGF transfection in KLM-1 cells reduced VEGF-A expression in the cells. KGFR and KGF may act to promote venous invasion and tumor angiogenesis in PDAC, raising the possibility that they may serve as novel therapeutic targets in anti-angiogenic strategies in PDAC.

Advanced glycation end products (AGEs) form non-enzymatically from reactions of proteins with reducing sugars. In the skin, AGEs were reported to accumulate in dermal elastin and collagens and to interact nonspecifically with the cell membrane of dermal fibroblasts. Therefore, AGEs may influence the process of skin aging. We investigated the presence of the AGE receptor RAGE in skin and the influence of AGEs on receptor expression and the formation of extracellular matrix (ECM). Sections of sun-protected and sun-exposed skin were analyzed with monoclonal antibodies against (RAGE), heat-shock protein 47, <em>factor</em> XIIIa, CD31, and CD45. RAGE was mainly expressed in fibroblasts, dendrocytes, and <em>keratinocytes</em> and to a minor extent in endothelial and mononuclear cells. Human foreskin fibroblasts (HFFs) highly expressed RAGE on the protein and mRNA level when analyzed by quantitative Western blotting and real-time PCR. Incubation of HFFs with the specific RAGE ligand Nepsilon-(carboxymethyl)lysine-modified BSA (CML-BSA) and tumor necrosis <em>factor</em>-alpha resulted in significant upregulation of RAGE expression. CML-BSA induced a mildly profibrogenic pattern, increasing connective tissue <em>growth</em> <em>factor</em>, transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) 1, and procollagen-alpha1(I) mRNA, whereas expression of matrix metalloproteinase (MMP)-1, -<em>2</em>, -3, and -1<em>2</em> was unaffected. We conclude that in HFFs, AGE-RAGE interactions may influence the process of skin aging through mild stimulation of ECM gene expression.

OBJECTIVE

To evaluate the safety of recombinant human keratinocyte growth factor (KGF) when administered with fluorouracil (FU) in patients with metastatic colorectal cancer.

METHODS

Patients (N = 81) received KGF by intravenous (IV) bolus on days 1 to 3, followed by FU 425 mg/m2/d IV bolus plus leucovorin 20 mg/m2/d IV on days 4 to 8. KGF dose levels were 1, 10, 20, 40, 60, and 80 microg/kg/d. A randomized, placebo-controlled design was employed (2:1 randomization of KGF to placebo). Oral mucositis was assessed by examination on days 1, 4, 8, 15, and 28. In addition, patients provided daily assessments of oral symptoms using a self-administered questionnaire.

RESULTS

Skin and oral events occurred in 13 of 18 patients (eight patients, grade 1; four patients, grade 2; and one patient, grade 3) treated with 60 and 80 microg/kg of KGF and three of 11 patients treated with 40 microg/kg (grade 1). These symptoms were dose limiting in three cases (ie, in two of 10 patients treated with 80 microg/kg and in one of eight patients treated with 60 microg/kg). The frequency of grade 2 to 4 mucositis was 43% in patients treated with KGF, compared with 67% in patients treated with placebo (P =.06). Patient self-assessments of oral pain and clinical assessments of mucositis showed good correlation (Kendall's tau = 0.75).

CONCLUSIONS

KGF is generally well tolerated when administered IV at doses up to 40 microg/kg/d for 3 days before a 5-day course of FU plus leucovorin. A clinically meaningful biologic effect was also suggested in that patients treated with the epithelial growth factor KGF had a lower rate of grade 2 to 4 mucositis than did patients treated with placebo.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has been used successfully to prevent alveolar damage induced by oxygen exposure in rodents. However, this treatment was used intratracheally and before oxygen exposure, which limited its clinical application. In the present study, mice were treated with the recombinant human KGF intravenously before (days -<em>2</em> and -1) or during (days 0 and +1) oxygen exposure. In both cases, lung damage was attenuated. KGF increased the number of cells incorporating bromodeoxyuridine (BrdU) in the septa and in bronchial epithelium of air-breathing mice but not of oxygen-exposed mice, indicating that the protective effect of KGF is not necessarily associated with proliferation. Oxygen-induced damage of alveolar epithelium and, unexpectedly, of endothelium was prevented by KGF treatment as seen by electron microscopy. We investigated the effect of KGF on different mechanisms known to be involved in oxygen toxicity. The induction of p53, Bax, and Bcl-x mRNAs during hyperoxia was to a large extent prevented by KGF. Surfactant proteins A and B mRNAs were not markedly modified by KGF. The anti-fibrinolytic activity observed in the alveoli during hyperoxia was to a large extent prevented by KGF, most probably by suppressing the expression of plasminogen activator inhibitor-1 (PAI-1) mRNA and protein. As PAI-1 -/- mice are more resistant to hyperoxia, KGF might act, at least in part, by decreasing the expression of this protease inhibitor and by restoring the fibrinolytic activity into the lungs.

Rat mucosal <em>keratinocytes</em> serially propagated under permanently serum-free conditions responded to interleukin (IL)-1 beta/IL-alpha and to transforming <em>growth</em> <em>factor</em> (TGF)-alpha/epidermal <em>growth</em> <em>factor</em> (EGF) (as well as to 1<em>2</em>-O-tetradecanoylphorbol-13-acetate (TPA)) by upregulation of M(r) 95,000 gelatinase (MMP-9) (M(r) 95K GL) and fibroblast-type collagenase (MMP-1) (FIB-CL), whereas control cells expressed barely detectable levels of either of these enzymes. The cells secreted 8-10 micrograms/10(6) cells/day (M(r) 95K GL) and <em>2</em>-3 micrograms/10(6) cells/day (FIB-CL) of enzyme protein for at least <em>2</em>4 h when maximally induced. This level was attained only after a <em>2</em>4-h lag period, and the earliest emergence of enzyme protein in the culture medium required 10-14 h. IL-1 beta was by far the most potent cytokine with maximal effect already at 10(-10) M, whereas IL-1 alpha, TGF-alpha, and EGF required <em>2</em>0-100-fold higher concentrations. Pretreatment of the cells with TPA (10(-7) M) abolished the subsequent response to IL-1 beta, TGF-alpha, and EGF and at the same time resulted in>> 90% reduction of cytosolic protein kinase C activity. Surprisingly, staurosporine, a potent kinase inhibitor, not only failed to block <em>growth</em> <em>factor</em>/cytokine responses but itself stimulated expression of the enzymes at a magnitude comparable to TPA. The inducing effect of TGF-alpha/EGF was down-regulated by 70-85% by 10(-7) M dexamethasone. Dexamethasone was less effective in ablating the IL-1 beta response yielding 60% reduction M(r) 95K GL and little or no reduction of FIB-CL. Dexamethasone also failed to block the TPA response.

Glutamine (Gln) and <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) each stimulate intestinal epithelial cell <em>growth</em>, but regulatory mechanisms are not well understood. We determined whether Gln and KGF alter intra- and extracellular thiol/disulfide redox pools in Caco-<em>2</em> cells cultured in oxidizing or reducing cell medium and whether such redox variations are a determinant of proliferative responses to these agents. Cells were cultured over a physiological range of oxidizing to reducing extracellular thiol/disulfide redox (Eh) conditions, obtained by varying cysteine (Cys) and cystine (CySS) concentrations in cell medium. Cell proliferation was determined by 5-bromo-<em>2</em>-deoxyuridine (BrdU) incorporation. Gln (10 mmol/l) or KGF (10 microg/l) did not alter BrdU incorporation at reducing Eh (-131 to -150 mV), but significantly increased incorporation at more oxidizing Eh (Gln at 0 to -109 mV; KGF at -46 to -80 mV). Cellular glutathione/glutathione disulfide (GSH/GSSG) Eh was unaffected by Gln, KGF, or variations in extracellular Cys/CySS Eh. Control cells largely maintained extracellular Eh at initial values after <em>2</em>4 h (-36 to -136 mV). However, extracellular Eh shifted toward a narrow physiological range with Gln and KGF treatment (Gln -56 to -88 mV and KGF -76 to -9<em>2</em> mV, respectively; P < 0.05 vs. control). The results indicate that thiol/disulfide redox state in the extracellular milieu is an important determinant of Caco-<em>2</em> cell proliferation induced by Gln and KGF, that this control is independent of intracellular GSH redox status, and that both Gln and KGF enhance the capability of Caco-<em>2</em> cells to modulate extremes of extracellular redox.

Exposure to the sun's UV radiation appears to be the most important environmental <em>factor</em> involved in the development of skin cancer. UVA is the major portion of UV radiation in sunlight and is considered to be a human carcinogen. In this study, we have investigated the delayed and sustained activation of ERK MAPK by UVA exposure. In parallel, a delayed Ras activation with a similar time course was observed after UVA exposure. The activated Ras was found to be localized in endomembranes such as the Golgi apparatus instead of plasma membranes. Expression of dominant negative Ras (N17Ras) abolished ERK activation by UVA. The presence of AG1478, an epidermal <em>growth</em> <em>factor</em> (EGF) receptor (EGFR) kinase inhibitor, had no effect on ERK or Ras activation, indicating that EGFR kinase activity is not involved in ERK activation by UVA. In contrast, protein kinase C (PKC) depletion by chronic 1<em>2</em>-O-tetradecanoylphorbol-13-acetate treatment nearly abolished UVA-induced ERK and Ras activation. The presence of the Ca(<em>2</em>+)-dependent-PKC inhibitor Go6976 had a similar effect. These findings suggest that ERK activation by UVA is mediated by PKC in a Ras-dependent pathway. In addition, a gradual increase in intracellular calcium level after UVA exposure was detected by flow cytometry. The presence of the PLC inhibitor U731<em>2</em><em>2</em> or the calcium chelator 1,<em>2</em>-bis(<em>2</em>-aminophenoxy)ethane-N,N, N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM) blocked both ERK and Ras activation, suggesting that both PLC and calcium are required for ERK activation. Our findings demonstrated that, different from UVC and UVB, UVA-induced delayed and sustained ERK activation is EGFR kinase activity-independent, but PLC/calcium/PKC-mediated. The delayed and sustained ERK activation provides a survival signal to human HaCaT <em>keratinocytes</em>, which may serve as an important mechanism for cell transformation and potential skin carcinogenesis in vivo caused by UVA exposure.

Previous work has shown that the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) tyrosine kinase moiety provides protection to normal human <em>keratinocytes</em> against apoptosis. This protection is, at least in part, due to EGFR-dependent expression of the antiapoptotic Bcl-<em>2</em> family member, Bcl-x(L). Here we focused on intracellular signaling pathways relevant to <em>keratinocyte</em> survival and/or Bcl-x(L) expression. By using pharmacological inhibitors and dominant negative expression constructs, we observed that phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha activation were required for <em>keratinocyte</em> survival independently of EGFR activation or Bcl-x(L) expression. By contrast, MEK activity required EGFR activation and, as shown by use of the MEK inhibitor PD98059 and a dominant negative MEK construct, was necessary for Bcl-x(L) expression and survival. Consistent with an earlier study, blocking SRC kinase activities similarly led to down-regulation of Bcl-x(L) protein expression and impaired <em>keratinocyte</em> survival. In conclusion, our results demonstrate that EGFR-dependent MEK activity contributes to both Bcl-x(L) expression and survival of normal <em>keratinocytes</em>. Other signaling pathways (i.e. phosphatidylinositol 3-kinase/AKT and phospholipase C gamma/protein kinase C alpha) are obligatory to <em>keratinocyte</em> survival but not to Bcl-x(L) expression, and control of these pathways by EGFR activation is not rate-limiting to normal <em>keratinocyte</em> survival.

Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (<em>2</em>5 ml/kg) with hyperoxia (50% O(<em>2</em>)) in rats to investigate alveolar type II (AT<em>2</em>) cell adhesion and focal adhesion signaling. AT<em>2</em> cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of focal adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of p130(Cas). RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT<em>2</em> cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT<em>2</em> cells that may be deleterious for cell adhesion and migration.

Members of the transforming <em>growth</em> <em>factor</em> beta/bone morphogenetic protein (TGF-beta/BMP) family are involved in the control of hair follicle (HF) morphogenesis and cycling. The activities of several members of this family activins and BMP-<em>2</em>, -4, -7, and -11) are controlled by antagonists such as follistatin. Because follistatin-deficient mice show abnormalities in vibrissae development, we explored the role of follistatin and activin in pelage HF development and cycling. We show here that during HF development follistatin mRNA was prominently expressed by hair matrix and outer root sheath <em>keratinocytes</em> as well as by interfollicular epidermal cells, whereas activin betaA mRNA was mainly expressed in dermal papilla cells. Compared with age-matched wild-type controls, both follistatin knockout mice and activin betaA transgenic mice showed a significant retardation of HF morphogenesis. Treatment of wild-type embryonic skin explants with follistatin protein stimulated HF development. This effect was inhibited by addition of recombinant activin A protein. Activin betaA transgenic mice demonstrated retardation of catagen entry, down-regulation of BMP-<em>2</em>, and up-regulation of expression of its antagonist matrix GLA protein. These observations suggest that follistatin and activin interaction plays an important role in both HF development and cycling, possibly in part by regulating expression of BMP-<em>2</em> and its antagonist.

Head and neck squamous cell carcinomas (HNSCCs) are associated with abnormal cell-mediated immunity at the primary tumor site. We investigated tumor-derived cytokines as <em>factors</em> underlying such abnormalities. Cytokine mRNA and protein of eight HNSCC-derived cell lines were tested; reverse transcription-PCR results indicated the presence of mRNAs for interleukin 1alpha (IL-1alpha) and transforming <em>growth</em> <em>factor</em> alpha (8 of 8); transforming <em>growth</em> <em>factor</em> beta and IL-1beta (7 of 8); and IL-4 and IL-6 (4 of 8). IL-<em>2</em>, IFN-gamma, and tumor necrosis <em>factor</em> alpha mRNA were not detected. Supernatants from six of these cell lines were analyzed by ELISA; IL-1alpha, IL-1beta, and IL-6 were found to be markedly increased compared to human papillomavirus-16-immortalized human oral <em>keratinocytes</em>. To determine whether the cell line findings are applicable to primary tumors, we performed immunohistochemical analysis on tumor specimens from 1<em>2</em> patients with invasive HNSCC. Universal intracellular production of IL-1alpha, IL-1beta, and IL-6 protein was detected. We conclude that the aberrant elaboration of biologically active IL-1 and IL-6 may contribute to altered immune status in HNSCC patients.

OBJECTIVE

Salivary glands are significantly affected when head and neck cancer patients are treated by radiation. We evaluated the effect of human keratinocyte growth factor (hKGF) gene transfer to murine salivary glands on the prevention of radiation-induced salivary hypofunction.

METHODS

A hybrid serotype 5 adenoviral vector encoding hKGF (AdLTR(2)EF1α-hKGF) was constructed. Female C3H mice, 8 weeks old, were irradiated by single (15 Gy) or fractionated (6 Gy for 5 days) doses to induce salivary hypofunction. AdLTR(2)EF1α-hKGF or AdControl was administered (10(8) - 10(10) particles per gland) to both submandibular glands (SG) by retrograde ductal instillation before irradiation (IR). Salivary flow was measured following pilocarpine stimulation. Human KGF levels were measured by ELISA. SG cell proliferation was measured with bromodeoxyuridine labeling. Endothelial and progenitor or stem cells in SGs were measured by flow cytometry. The effect of SG hKGF production on squamous cell carcinoma (SCC VII) tumor growth was assessed.

RESULTS

In 3 separate single-dose IR experiments, salivary flow rates of mice administered the AdLTR(2)EF1α-hKGF vector were not significantly different from nonirradiated control mice (P>> 0.05). Similarly, in 3 separate fractionated IR experiments, the hKGF-expressing vector prevented salivary hypofunction dramatically. Transgenic hKGF protein was found at high levels in serum and SG extracts. AdLTR(2)EF1α-hKGF-treated mice showed increased cell proliferation and numbers of endothelial cells, compared with mice treated with AdControl. hKGF gene transfer had no effect on SCC VII tumor growth ± radiation.

CONCLUSIONS

hKGF gene transfer prevents salivary hypofunction caused by either single or fractionated radiation dosing in mice. The findings suggest a potential clinical application.

The angiogenic cytokine vascular endothelial <em>growth</em> <em>factor</em> (VEGF)-A plays a central role in both wound healing and tumor <em>growth</em>. In the skin, epidermal <em>keratinocytes</em> are a major source of this <em>growth</em> <em>factor</em>. To study the contribution of <em>keratinocyte</em>-derived VEGF-A to these angiogenesis-dependent processes, we generated mice in which this cytokine was inactivated specifically in keratin 5-expressing tissues. The mutant mice were macroscopically normal, and the skin capillary system was well established, demonstrating that <em>keratinocyte</em>-derived VEGF-A is not essential for angiogenesis in the skin during embryonic development. However, healing of full-thickness wounds in adult animals was appreciably delayed compared with controls, with retarded crust shedding and the appearance of a blood vessel-free zone underneath the newly formed epidermis. When 9,1<em>2</em>-dimethyl 1,<em>2</em>-benzanthracene was applied as both tumor initiator and promoter, a total of 143 papillomas developed in <em>2</em>0 of <em>2</em>3 (87%) of control mice. In contrast, only three papillomas arose in <em>2</em> of 17 (1<em>2</em>%) of the mutant mice, whereas the rest merely displayed epidermal thickening and parakeratosis. Mutant mice also developed only <em>2</em> squamous cell carcinomas, whereas 11 carcinomas were found in seven of the control animals. These data demonstrate that whereas <em>keratinocyte</em>-derived VEGF-A is dispensable for skin vascularization under physiological conditions, it plays an important albeit nonessential role during epidermal wound healing and is crucial for the development of 9,1<em>2</em>-dimethyl 1,<em>2</em>-benzanthracene-induced epithelial skin tumors.

The human papillomavirus type 16 (HPV16) E5 protein associates with the epidermal <em>growth</em> <em>factor</em> receptor (EGFR) and enhances the activation of the EGFR after stimulation by EGF in human <em>keratinocytes</em>. Phosphatidylinositol 3-kinase (PI3K) and ERK1/<em>2</em> mitogen-activated protein kinase (ERK1/<em>2</em> MAPK), two signal molecules downstream of the EGFR, have been recognized as participants in two survival signal pathways in response to stress. The fact that E5 can enhance EGFR activation suggests that E5 might act as a survival <em>factor</em>. To test this hypothesis, the apoptotic response of UV B-irradiated primary <em>keratinocytes</em> infected with either control retrovirus, LXSN, or HPV16 <em>2</em>E5-expressing recombinant retrovirus was quantitated. Under the same conditions, LXSN-infected cells showed extensive apoptosis, while E5-expressing cells demonstrated a significant reduction in UV B-irradiation-induced apoptosis. The E5-mediated protection against apoptosis was blocked by wortmannin and PD98059, specific inhibitors of the PI3K and ERK1/<em>2</em> MAPK pathways, respectively, suggesting that the PI3K and ERK1/<em>2</em> MAPK pathways are involved in this process. Western blot analysis showed that Akt (also named protein kinase B), which is a downstream effector of PI3K, and ERK1/<em>2</em> MAPK were activated by EGF. When cells were stimulated by EGF and irradiated by UV B, the levels of phospho-Akt and phospho-ERK1/<em>2</em> activated by EGF in E5-expressing cells were about twofold greater than those in LXSN-infected cells. Two other UV-activated stress pathways, p38 and JNK, were activated to the same level during UV B irradiation in both LXSN-infected cells and E5-expressing cells, indicating that E5 protein did not affect these two pathways. After UV B irradiation, p53 was activated in both LXSN-infected cells and E5-expressing cells, and cell cycle analysis showed that nearly all cells in both cell populations were <em>growth</em> arrested. These data suggest that unlike HPV16 E6, which blocks apoptosis by inactivation of p53, HPV16 E5 protects cells from apoptosis by enhancing the PI3K-Akt and ERK1/<em>2</em> MAPK signal pathways.

Tissue remodeling follows the initial phase of wound healing and stops inflammatory and scar-forming processes, then restores the normal tissue morphology. The human peptide Gly-(L-His)-(L-Lys) or GHK, has a copper <em>2</em>+ (Cu(<em>2</em>+)) affinity similar to the copper transport site on albumin and forms GHK-Cu, a complex with Cu(<em>2</em>+). These two molecules activate a plethora of remodeling related processes: (1) chemoattraction of repair cells such as macrophages, mast cells, capillary cells; (<em>2</em>) anti-inflammatory actions (suppression of free radicals, thromboxane formation, release of oxidizing iron, transforming <em>growth</em> <em>factor</em> beta-1, tumor necrosis <em>factor</em> alpha and protein glycation while increasing superoxide dismutase, vessel vasodilation, blocking ultraviolet damage to skin <em>keratinocytes</em> and improving fibroblast recovery after X-ray treatments); (3) increases protein synthesis of collagen, elastin, metalloproteinases, anti-proteases, vascular endothelial <em>growth</em> <em>factor</em>, fibroblast <em>growth</em> <em>factor</em> <em>2</em>, nerve <em>growth</em> <em>factor</em>, neutrotropins 3 and 4, and erythropoietin; (4) increases the proliferation of fibroblasts and <em>keratinocytes</em>; nerve out<em>growth</em>, angiogenesis, and hair follicle size. GHK-Cu stimulates wound healing in numerous models and in humans. Controlled studies on aged skin demonstrated that it tightens skin, improves elasticity and firmness, reduces fine lines, wrinkles, photodamage and hyperpigmentation. GHK-Cu also improves hair transplant success, protects hepatic tissue from tetrachloromethane poisoning, blocks stomach ulcer development, and heals intestinal ulcers and bone tissue. These results are beginning to define the complex biochemical processes that regulate tissue remodeling.

Human prostate is now considered a target for vitamin D receptor (VDR) ligands, such as BXL-6<em>2</em>8. Because BXL-6<em>2</em>8 inhibited prostate <em>growth</em> without interfering with androgen signaling, it represents a new option for benign prostate hyperplasia (BPH) therapy. However, BPH symptoms are related not only to prostate size, but also to compensatory bladder hypertrophy and eventual overactivity. We now report that human bladder expresses VDR (determined by real-time PCR immunohistochemistry and Western blot) and responds to VDR agonists, such as the natural ligand, calcitriol, and its synthetic and less hypercalcemic derivative, BXL-6<em>2</em>8. Experiments were conducted with stromal cells derived from human bladder neck obtained at surgery from BPH patients. BXL-6<em>2</em>8 counteracted <em>keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) and androgen-induced cell proliferation and stimulated apoptosis with a parallel reduced expression of the survival oncoprotein Bcl-<em>2</em>. Prolonged serum starvation time-dependently pushed bladder stromal cells to express activated myofibroblast markers, such as desmin and smoothelin, without changing other contractile-related proteins and intermediate filaments, such as vimentin. Chronic exposure to BXL-6<em>2</em>8 prevented starvation-induced cell phenotype modification. Because hypertrophy and starvation-induced bladder remodeling are supposed to underlie bladder overactivity, it is possible that BXL-6<em>2</em>8 might be helpful in reducing not only cumbersome symptoms related to prostate over<em>growth</em>, but also those related to bladder irritation.

This paper identifies a new, developmental role for transcription <em>factor</em> AP-<em>2</em> in the activation of amphibian embryonic epidermal keratin gene expression. Keratin transcription <em>factor</em> KTF-1 is shown by several criteria to be identical or closely related to AP-<em>2</em>. KTF-1/AP-<em>2</em> is shown to be tissue-specific from its first transcription in Xenopus embryos, and restricted to a small number of adult tissues, including skin. Epidermis-specific keratin transcription closely follows specification of the embryonic ectoderm in Xenopus, and is subject to regulation by <em>growth</em> <em>factors</em> and embryonic induction. We further show that in mouse basal <em>keratinocytes</em>, a KTF-1/AP-<em>2</em>-like <em>factor</em> is present and binds to a DNA sequence previously shown to be important in the regulation of the keratin K14 gene, which is actively expressed in these cells. Thus, the study of AP-<em>2</em> and its role in the regulation of keratin gene transcription should enhance our understanding of both amphibian embryonic development and mammalian skin differentiation.

<em>Growth</em> <em>factors</em> and <em>growth</em> <em>factor</em> receptors are involved in tumor progression. The fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> gene encodes distinct isoforms. The isoforms which bind KGF (<em>keratinocyte</em> <em>growth</em> <em>factor</em> or FGF-7) are called KGF-R or FGFR<em>2</em>b. KGF-R is expressed in different epithelia and is involved in the control of epithelial-mesenchymal interactions. Expression of KGF-R mRNA was examined in normal human bladder and transitional cell carcinoma of the bladder (TCC) by semi-quantitative RT-PCR using TFIID and GAPDH as internal standards. In normal bladder, the KGF-R mRNA was detected in the urothelium but not in the underlying stroma. In TCCs, the level of KGF-R mRNA was generally either normal or low. Eighteen out of 54 TCCs had a KGF-R mRNA level below 30% of that found in normal urothelium. This decrease in KGF-R mRNA was not accompanied by an increase in BEK (FGFR<em>2</em>c) mRNA, the other major splice variant of the fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> gene. Expression of the KGF-R was also monitored by immunohistochemistry using a functional KGF-immunoglobulin chimera. The receptor was uniformly expressed throughout the normal urothelium except for the umbrella cells. Immunoreactivity for KGF-R was found to be negative in tumors with low levels of KGF-R mRNA, while the peritumoral normal urothelium was positive. Among patients with muscle invasive tumors, those exhibiting a low level of KGF-R mRNA had a significantly higher proportion of cancer deaths. Our results suggest that decreased expression of KGF-R can be considered as a marker of tumor progression in muscle invasive TCCs.

<em>Growth</em> <em>factors</em>, including fibroblast <em>growth</em> <em>factor</em>-<em>2</em> (FGF-<em>2</em>) and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) regulate fibroblast function, differentiation and proliferation. S100A8 and S100A9 are members of the S100 family of Ca<em>2</em>+-binding proteins and are now accepted as markers of inflammation. They are expressed by <em>keratinocytes</em> and inflammatory cells in human/murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and <em>keratinocytes</em> in vitro. In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts. Endotoxin (LPS), interferon gamma (IFNgamma), tumour-necrosis <em>factor</em> (TNF) and TGF-beta did not induce the S100A8 gene in murine fibroblasts whereas FGF-<em>2</em> induced mRNA maximally after 1<em>2</em> h. The FGF-<em>2</em> response was strongly enhanced and prolonged by heparin. Interleukin-1beta (IL-1beta) alone, or in synergy with FGF-<em>2</em>/heparin strongly induced the gene in 3T3 fibroblasts. S100A9 mRNA was not induced under any condition. Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts. S100A8 mRNA induction by FGF-<em>2</em> and IL-1beta was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis. FGF-<em>2</em>-responsive elements were distinct from the IL-1beta-responsive elements in the S100A8 gene promoter. FGF-<em>2</em>-/heparin-induced, but not IL-1beta-induced responses were significantly suppressed by TGF-beta, possibly mediated by decreased mRNA stability. S100A8 in activated fibroblasts was mainly intracytoplasmic. Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells <em>2</em> and 4 days post injury; numbers declined by 7 days. Up-regulation of S100A8 by FGF-<em>2</em>/IL-1beta, down-regulation by TGF-beta, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair.

AP-1 transactivation appears to be required for mouse JB6 cell neoplastic transformation induced by the tumor promoter TPA or epidermal <em>growth</em> <em>factor</em> (EGF). Exposure to AP-1 transrepressing retinoids and glucocorticoids and expression of a dominant negative c-jun (TAM67) blocked tumor promoter-induced AP-1 transactivation and neoplastic transformation. The aim of the present study was to extend the inquiry of the role of AP-1 and other transcription <em>factors</em> to human neoplastic progression. Expression of human papillomavirus (HPV) 16 or 18 E6 and E7 immortalizes human <em>keratinocytes</em> and inhibits serum/calcium-stimulated differentiation. Further transformation by v-fos co-expression renders these <em>keratinocytes</em> tumorigenic in nude mice. We have analysed two series of E6/E7 immortalized human <em>keratinocyte</em> cell lines that show progressing phenotypes ranging from differentiation sensitive to anchorage-independent to tumorigenic in nude mice. We analysed the activities of AP-1 and NF-kappaB which may 'cross-talk'. Both DNA binding and transactivation of AP-1 and NF-kappaB transcription <em>factors</em> showed elevation in the anchorage-independent (16RH) and tumorigenic (18 v-fos) <em>keratinocyte</em> lines compared to the less progressed but immortalized cell lines. HPV E7 was expressed at a constant level shown by quantitative RT-PCR in both the more and the less progressed lines, indicating that E7 is not the <em>factor</em> limiting this progression. Blocked shift/supershift analysis indicates that Fos family member proteins especially Fra-1 and Fra-<em>2</em> are related to progression and no changes found in the Jun family member proteins although they are present in the AP-1/DNA binding complex. When a dominant negative mutant c-jun driven by a human keratin 14 promoter was co-transfected with AP-1 or NF-kappaB reporters, both AP-1 and NF-kappaB activities were suppressed in the more progressed cell lines 16RH and 18 v-fos but not in the less progressed 16RL or 18 cell lines. Overexpression of the same dominant negative c-jun did not inhibit p53 dependent reporter transactivation, indicating the specificity of inhibition of AP-1 and NF-kappaB transactivation in the HPV-immortalized cells. Stable transfectants of this mutant c-jun in the two more progressed cell lines 16RH and 18 v-fos showed reduced AP-1 and NF-kappaB activation and reduced anchorage-independent <em>growth</em>. Together, these results indicate that activation of AP-1, NF-kappaB or both may contribute to neoplastic progression in HPV immortalized human <em>keratinocytes</em> and that specific targeting of the elevated levels seen in benign or malignant tumors might be effective for prevention or treatment of human cancer.

The survival of type <em>2</em> alveolar epithelial cells (AEC<em>2</em>) in the lung after hyperoxic injury is regulated by signals from the cellular environment. <em>Keratinocyte</em> <em>growth</em> <em>factor</em> and Matrigel can ameliorate the hallmarks of apoptosis seen in hyperoxic AEC<em>2</em> after <em>2</em>4-h culture on plastic [S. Buckley, L. Barsky, B. Driscoll, K. Weinberg, K. D. Anderson, and D. Warburton. Am. J. Physiol. <em>2</em>74 (Lung Cell. Mol. Physiol. 18): L714-L7<em>2</em>0, 1998]. We used the same model of in vivo short-term hyperoxia to characterize the protective effects of substrate attachment. Culture of hyperoxic AEC<em>2</em> on various biological adhesion substrates showed reduced DNA end labeling in cells grown on all biological substrates compared with <em>growth</em> on plastic. In contrast, the synthetic substrate poly-D-lysine conferred no protection. Hyperoxic AEC<em>2</em> cultured on laminin showed an increased ratio of expression of Bcl-<em>2</em> to interleukin-1beta-converting enzyme compared with culture on plastic. Laminin also partially restored hyperoxia-depleted glutathione levels and conferred improved optimal mitochondrial viability as measured by the 3-(4,5-dimethylthiazol-<em>2</em>-yl)-<em>2</em>,5-diphenyltetrazolium bromide (MTT) assay. Conversely, attachment to the nonphysiological substrate poly-D-lysine afforded no such protection, suggesting that protection against hyperoxia-induced damage may be associated with integrin signaling. Increased activation of extracellular signal-regulated kinase (ERK), as detected by increased ERK tyrosine phosphorylation, was seen in hyperoxic AEC<em>2</em> as soon as the cells started to attach to laminin and was sustained after <em>2</em>4 h of culture in contrast to that in control AEC<em>2</em>. To confirm that protection against DNA strand breakage and apoptosis was being conferred by ERK activation, the cells were also plated in the presence of 50 microM PD-98059, an inhibitor of the ERK-activating mitogen-activating kinase. Culture for <em>2</em>4 h with PD-98059 abolished the protective effect of laminin. We speculate that after hyperoxic lung injury, signals through the basement membrane confer specific protection against oxygen-induced DNA strand breakage and apoptosis through an ERK activation-dependent pathway.

The human papillomavirus type 16 (HPV-16), the type most often associated with cervical cancer, immortalizes primary <em>keratinocytes</em> and inhibits serum/calcium-stimulated differentiation in culture. In this study, we have used a model of <em>keratinocyte</em> immortalization based upon HPV-16 to analyze perturbation of function and expression of E-cadherin, a Ca(<em>2</em>+)-dependent cell-cell adhesion molecule expressed by normal <em>keratinocytes</em>, and its associated proteins. An immortalized <em>keratinocyte</em> cell line generated by cotransfection with HPV-16 E6 and E7 showed decreased membrane E-cadherin expression and redistribution of alpha-, beta-, and gamma-catenin from the undercoat membrane to the cytoplasm. No changes in the level of expression were seen. Selection of the immortalized <em>keratinocyte</em> cell line for resistance to differentiation generated a more transformed cell line with an invasive phenotype, down-regulated E-cadherin and alpha-catenin, and up-regulated the epidermal <em>growth</em> <em>factor</em> receptor (EGFr). Transfection of an E-cadherin expression construct into the differentiation-resistant cell line restored membrane-bound E-cadherin and catenin expression, down-regulated the EGFr, and reversed the invasive phenotype. These results indicate that overexpression of the EGFr correlates with perturbation of the E-cadherin/catenin complex seen in the HPV-16 E6- and E7-transfected <em>keratinocytes</em> and may underlie a functional interaction between <em>growth</em>-regulatory <em>factors</em> and adhesion molecules (E-cadherin/catenin).

Fetal mesenchyme-derived <em>factors</em> are likely to play an important role in pancreatic islet development and <em>growth</em>. We have used primary cultures of human fetal pancreatic tissue to identify <em>growth</em> <em>factors</em> that have morphogenic, mitogenic, and insulinotropic activity. The formation of islet-like cell clusters (ICCs) during a 6-day culture was stimulated two- to threefold by hepatocyte <em>growth</em> <em>factor</em>/scatter <em>factor</em> (HGF/SF) basic fibroblast <em>growth</em> <em>factor</em> (FGF)-<em>2</em>, and to a lesser extent by <em>keratinocyte</em> <em>growth</em> <em>factor</em> (FGF-7) and insulin-like <em>growth</em> <em>factor</em>-II (IGF-II). In contrast, transforming <em>growth</em> <em>factor</em>-beta (TGF-beta) had a strong inhibitory effect. The ICCs formed during HGF/SF stimulation consisted mainly of epithelial cells, whereas FGF-<em>2</em>-induced ICCs were predominantly nonepithelial. Furthermore, although both FGF-<em>2</em> and HGF/SF increased the total insulin content of the cultures, only HGF/SF increased the insulin content per DNA. Quantitatively, HGF/SF stimulated a <em>2</em>.3-fold increase in the proportion of insulin-positive cells and a 3-fold higher number of replicating beta-cells. Blocking of the IGF-I receptor inhibited ICC formation but did not affect their insulin content. Immunoneutralizing TGF-beta resulted in increased cell <em>growth</em> and insulin content, indicating the presence of an endogenous inhibitory TGF-beta activity in the model system. Our results suggest that HGF/SF may be an important component of the fetal mesenchyme-derived <em>factors</em> responsible for pancreatic islet development. HGF/SF also may prove valuable for supporting the in vitro <em>growth</em> of islet cells.

<em>Keratinocyte</em> <em>growth</em> <em>factor</em> (KGF) has been implicated in wound re-epithelialization and branching morphogenesis of several organs. To determine whether KGF induces these effects via induction of matrix metalloproteinase expression we have analysed the effect of KGF on the expression of stromelysin-<em>2</em> in cultured HaCaT <em>keratinocytes</em>. Here we show a strong induction of stromelysin-<em>2</em> mRNA within 5-8 h of stimulation of these cells with KGF. The degree of induction was similar to that achieved by treatment with epidermal <em>growth</em> <em>factor</em> or tumour necrosis <em>factor</em> alpha, whereas the stimulatory effect of transforming <em>growth</em> <em>factor</em> beta 1 was even stronger. To determine whether the induction of stromelysin-<em>2</em> expression by <em>growth</em> <em>factors</em> and cytokines might be important for wound healing, we analysed the expression of this gene during the healing process of full-thickness excisional wounds in mice. Whereas stromelysin-<em>2</em> mRNA could hardly be detected in unwounded skin, a biphasic induction was seen after injury and highest levels were found at days 1 and 5 after wounding. Hybridization in situ revealed the presence of stromelysin-<em>2</em> mRNA in basal <em>keratinocytes</em> at the wound edge but not in the underlying mesenchymal tissue. During impaired wound healing as seen in glucocorticoid-treated mice, stromelysin-<em>2</em> expression was significantly increased compared with untreated control mice. Taken together, these results suggest that correct regulation of this broad-spectrum metalloproteinase might be important for normal repair.