We have studied the functional interaction between retinoic acid and transforming <em>growth</em> <em>factor</em>-beta (TGF-beta), using the mouse epidermis as a model system. Treatment with retinoic acid increases expression of TGF-beta <em>2</em> in cultured <em>keratinocytes</em> in vitro, as well as in the epidermis in vivo. This TGF-beta <em>2</em> is secreted in a biologically active form that can bind to surface receptors, in contrast to most other conditions in which TGF-beta is secreted in a latent form. Specific antibodies to TGF-beta <em>2</em> partially reverse the ability of retinoic acid to inhibit DNA synthesis in cultured <em>keratinocytes</em>. The regulation of TGF-beta <em>2</em> expression by retinoic acid may have important physiological and pharmacological roles in the maintenance of epidermal homeostasis.

Regulation of transforming <em>growth</em> <em>factor</em> beta 1 (TGF beta 1), TGF beta <em>2</em>, and TGF beta 3 mRNAs in murine fibroblasts and <em>keratinocytes</em> by TGF beta 1 and TGF beta <em>2</em> was studied. In quiescent AKR-<em>2</em>B fibroblasts, in which TGF beta induces delayed stimulation of DNA synthesis, TGF beta 1 autoregulation of TGF beta 1 expression was observed as early as 1 h, with maximal induction (<em>2</em>5-fold) after 6 to 1<em>2</em> h. Increased expression of TGF beta 1 mRNA was accompanied by increased TGF beta protein production into conditioned medium of AKR-<em>2</em>B cells. Neither TGF beta <em>2</em> nor TGF beta 3 mRNA, however, was significantly induced, but both were apparently down regulated at later times by TGF beta 1. Protein synthesis was not required for autoinduction of TGF beta 1 mRNA in AKR-<em>2</em>B cells. Nuclear run-on analyses and dactinomycin experiments indicated that autoregulation of TGF beta 1 expression is complex, involving both increased transcription and message stabilization. In contrast to TGF beta 1, TGF beta <em>2</em> treatment of quiescent AKR-<em>2</em>B cells increased expression of TGF beta 1, TGF beta <em>2</em>, and TGF beta 3 mRNAs, but with different kinetics. Autoinduction of TGF beta <em>2</em> mRNA occurred rapidly with maximal induction at 1 to 3 h, enhanced TGF beta 3 mRNA levels were observed after 3 h, and increased expression of TGF beta 1 occurred later, with maximal mRNA levels obtained after 1<em>2</em> to <em>2</em>4 h. Nuclear run-on analyses indicated that TGF beta <em>2</em> regulation of TGF beta <em>2</em> and TGF beta 3 mRNA levels is transcriptional, while TGF beta <em>2</em> induction of TGF beta 1 expression most likely involves both transcriptional and posttranscriptional controls. In BALB/MK mouse <em>keratinocytes</em>, minimal autoinduction of TGF beta 1 occurred at only the 1<em>2</em>- and <em>2</em>4-h time points and protein synthesis was required for this autoinduction. The results of this study provide an example in which TGF beta 1 and TGF beta <em>2</em> elicit different responses and demonstrate that expression of TGF beta 1, and TGF beta 3 are regulated differently. The physiological relevance of TGF beta 1 autoinduction in the context of wound healing is discussed.

1 Muscarinic M1-M5 receptors mediate the metabotropic actions of acetylcholine in the nervous system. A <em>growing</em> body of data indicate they also mediate autocrine functions of the molecule. The availability of novel and selective muscarinic agonists and antagonists, as well as in vivo gene disruption techniques, has clarified the roles of muscarinic receptors in mediating both functions of acetylcholine. <em>2</em> Selective M1 agonists or mixed M1 agonists/M<em>2</em> antagonists may provide an approach to the treatment of cognitive disorders, while M3 antagonism, or mixed M<em>2</em>/M3 antagonists, are approved for the treatment of contractility disorders including overactive bladder and chronic obstructive pulmonary disease. Preclinical data suggest that selective agonism of the M4 receptor will provide novel anti-nociceptive agents, while therapeutics-based upon agonism or antagonism of the muscarinic M5 receptor have yet to be reported. 3 The autocrine functions of muscarinic receptors broadly fall into two areas - control of cell <em>growth</em> or proliferation and mediation of the release of chemical mediators from epithelial cells, ultimately causing muscle relaxation. The former particularly are involved in embryological development, oncogenesis, <em>keratinocyte</em> function and immune responsiveness. The latter regulate contractility of smooth muscle in the vasculature, airways and urinary bladder. 4 Most attention has focused on muscarinic M1 or M3 receptors which mediate lymphocyte immunoresponsiveness, cell migration and release of smooth muscle relaxant <em>factors</em>. Muscarinic M4 receptors are implicated in the regulation of <em>keratinocyte</em> adhesion and M<em>2</em> receptors in stem cell proliferation and development. Little data are available concerning the M5 receptor, partly due to the difficulties in defining the subtype pharmacologically. 5 The autocrine functions of acetylcholine, like those in the nervous system, involve activation of several muscarinic receptor subtypes. Consequently, the role of these subtypes in autocrine, as well neuronal cholinergic systems, significantly expands their importance in physiology and pathophysiology.

The matrix metalloproteinases (MMPs) play a key role in normal and pathological angiogenesis by mediating extracellular matrix degradation and/or controlling the biological activity of <em>growth</em> <em>factors</em>, chemokines, and/or cytokines. Specific functions of individual MMPs as anti- or proangiogenic mediators remain to be elucidated. In the present study, we assessed the impact of single or combined MMP deficiencies in in vivo and in vitro models of angiogenesis (malignant <em>keratinocyte</em> transplantation and the aortic ring assay, respectively). MMP-9 was predominantly expressed by neutrophils in tumor transplants, whereas MMP-<em>2</em> and MMP-3 were stromal. Neither the single deficiency of MMP-<em>2</em>, MMP-3, or MMP-9, nor the combined absence of MMP-9 and MMP-3 did impair tumor invasion and vascularization in vivo. However, there was a striking cooperative effect in double MMP-<em>2</em>:MMP-9-deficient mice as demonstrated by the absence of tumor vascularization and invasion. In contrast, the combined lack of MMP-<em>2</em> and MMP-9 did not impair the in vitro capillary out<em>growth</em> from aortic rings. These results point to the importance of a cross talk between several host cells for the in vivo tumor promoting and angiogenic effects of MMP-<em>2</em> and MMP-9. Our data demonstrate for the first time in an experimental model that MMP-<em>2</em> and MMP-9 cooperate in promoting the in vivo invasive and angiogenic phenotype of malignant <em>keratinocytes</em>.

Apert syndrome, one of five craniosynostosis syndromes caused by allelic mutations of fibroblast <em>growth</em>-<em>factor</em> receptor <em>2</em> (FGFR<em>2</em>), is characterized by symmetrical bony syndactyly of the hands and feet. We have analyzed <em>2</em>60 unrelated patients, all but <em>2</em> of whom have missense mutations in exon 7, which affect a dipeptide in the linker region between the second and third immunoglobulin-like domains. Hence, the molecular mechanism of Apert syndrome is exquisitely specific. FGFR<em>2</em> mutations in the remaining two patients are distinct in position and nature. Surprisingly, each patient harbors an Alu-element insertion of approximately 360 bp, in one case just upstream of exon 9 and in the other case within exon 9 itself. The insertions are likely to be pathological, because they have arisen de novo; in both cases this occurred on the paternal chromosome. FGFR<em>2</em> is present in alternatively spliced isoforms characterized by either the IIIb (exon 8) or IIIc (exon 9) domains (<em>keratinocyte</em> <em>growth</em>-<em>factor</em> receptor [KGFR] and bacterially expressed kinase, respectively), which are differentially expressed in mouse limbs on embryonic day 13. Splicing of exon 9 was examined in RNA extracted from fibroblasts and <em>keratinocytes</em> from one patient with an Alu insertion and two patients with Pfeiffer syndrome who had nucleotide substitutions of the exon 9 acceptor splice site. Ectopic expression of KGFR in the fibroblast lines correlated with the severity of limb abnormalities. This provides the first genetic evidence that signaling through KGFR causes syndactyly in Apert syndrome.

Glucocorticoids (GCs) have a long history of use as therapeutic agents for numerous skin diseases. Surprisingly, their specific molecular effects are largely unknown. To characterize GC action in epidermis, we compared the transcriptional profiles of primary human <em>keratinocytes</em> untreated and treated with dexamethasone (DEX) for 1, 4, <em>2</em>4, 48, and 7<em>2</em> h using large scale microarray analyses. The majority of genes were found to be regulated only after <em>2</em>4 h and remained regulated throughout treatment. In addition to regulation of the expected pro-inflammatory genes, we found that GCs regulate cell fate, tissue remodeling, cell motility, differentiation, and metabolism. GCs suppress the expression of essentially all IFNgamma-regulated genes, including IFNgamma receptor and STAT-1, an effect that was previously unknown. GCs also block STAT-1 activation and nuclear translocation. Unexpectedly, GCs induce the expression of anti-apoptotic genes and repress pro-apoptotic ones, preventing UV-induced <em>keratinocyte</em> apoptosis. Consequently, treatment with GCs blocked UV-induced apoptosis of <em>keratinocytes</em>. GCs have profound effect on wound healing by inhibiting cell motility and the expression of the proangiogenic <em>factor</em>, vascular endothelial <em>growth</em> <em>factor</em>. They play an important role in tissue remodeling and scar formation by suppressing the expression of TGFbeta1 and -<em>2</em> and MMP1, -<em>2</em>, -9, and -10 and inducing TIMP-<em>2</em>. Finally, GCs promote terminal epidermal differentiation while simultaneously inhibiting early stage differentiation. These results provide new insights into the beneficial and adverse effects of GCs in the epidermis, defining the participating genes and mechanisms that coordinate the cellular responses important for GC-based therapies.

The fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em>-IIIb (FGFR<em>2</em>b) and the vascular endothelial <em>growth</em> <em>factor</em> receptor <em>2</em> (VEGFR<em>2</em>) are tyrosine kinases that can promote cell migration and proliferation and have important roles in embryonic development and cancer. Here we show that FGF7/FGFR<em>2</em>b-dependent activation of epidermal <em>growth</em> <em>factor</em> receptor (EGFR)/ERK1/<em>2</em> signalling and cell migration in epithelial cells require stimulation of the membrane-anchored metalloproteinase ADAM17 and release of heparin-binding epidermal <em>growth</em> <em>factor</em> (HB-EGF). Moreover, VEGF-A/VEGFR<em>2</em>-induced migration of human umbilical vein endothelial cells also depends on EGFR/ERK1/<em>2</em> signalling and shedding of the ADAM17 substrate HB-EGF. The pathway used by the FGF7/FGFR<em>2</em>b signalling axis to stimulate shedding of substrates of ADAM17, including ligands of the EGFR, involves Src, p38 mitogen-activated protein-kinase and PI3K, but does not require the cytoplasmic domain of ADAM17. Based on these findings, ADAM17 emerges as a central component in a triple membrane-spanning pathway between FGFR<em>2</em>b or VEGFR<em>2</em> and EGFR/ERK1/<em>2</em> that is required for cell migration in <em>keratinocytes</em> and presumably also in endothelial cells.

The visual appearance of humans derives predominantly from their skin and hair color. The phylogenetically ancient biochemical [corrected] pathway underling this phenomenon is called melanogenesis and results in the production of melanin pigments in neural crest-derived melanocytes, followed by its transfer to epithelial cells. While melanin from epidermal melanocytes clearly protects human skin by screening harmful ultraviolet radiation, the biologic value of hair pigmentation is less clear. In addition to important roles in social/sexual communication, one potential benefit of pigmented scalp hair in humans may be the rapid excretion of heavy metals, chemicals, toxins from the body by their selective binding to melanin. The hair follicle and epidermal melanogenic systems are broadly distinct, though open. The primary distinguishing feature of follicular melanogenesis, compared to the continuous melanogenesis in the epidermis, is the tight coupling of hair follicle melanogenesis to the hair <em>growth</em> cycle. This cycle appears to involve periods of melanocyte proliferation (during early anagen), maturation (mid to late anagen) and melanocyte death via apoptosis (during early catagen). Thus, each hair cycle is associated with the reconstruction of an intact hair follicle pigmentary unit... at least for the first 10 cycles or so. Thereafter, gray and white hairs appear, suggesting an age-related, genetically regulated exhaustion of the pigmentary potential of each individual hair follicle. Melanocyte aging may be associated with reactive oxygen species-mediated damage to nuclear and mitochondrial DNA with resultant accumulation of mutations with age, in addition to dysregulation of anti-oxidant mechanisms or pro/anti-apoptotic <em>factors</em> within the cells. While the perception of "gray hair" derives in large part from the admixture of pigmented and white hair, it is important to note that individual hair follicles can indeed exhibit pigment dilution or true grayness. This dilution is due to a reduction in tyrosinase activity of hair bulbar melanocytes, sub-optimal melanocyte-cortical <em>keratinocyte</em> interactions, and defective migration of melanocytes from a reservoir in the upper outer root sheath to the pigment-permitting microenvironment close to the dermal papilla of the hair bulb. Animal models with mutations in apoptotic survival <em>factors</em> (e.g. bcl-<em>2</em>) and in melanogenic enzymes (TRP-1) are providing valuable insights into the aging hair pigmentary unit. It is from these and other advances, including our ability to grow hair follicle melanocytes in vitro, that the possibility of reversing canities has been raised. Indeed, it is not too uncommon to see spontaneous repigmentation along the same individual hair shaft in early canities. Moreover, melanocytes taken from gray and white hair follicles can be induced to pigment in vitro. One of the surprising results of pigment loss in canities is the alteration in <em>keratinocyte</em> proliferation and differentiation, providing the tantalizing suggestion that melanocytes in the hair follicle contribute far more that packages of melanin alone. Furthermore, there have been some unconfirmed reports in the literature suggesting that canities may link (although not causally) with more systemic alterations in homeostasis e.g. osteoporosis. Here, we review the current state of knowledge of the development, regulation and control of the human hair follicle pigmentary unit during life.

MicroRNAs (miRNAs) are short, single-stranded, noncoding RNAs that play important roles in the regulation of gene expression. We previously identified a characteristic miRNA expression profile in psoriasis, distinct from that of healthy skin. One of the most downregulated miRNAs in psoriasis skin was microRNA-1<em>2</em>5b (miR-1<em>2</em>5b). In this study, we aimed to identify the potential role(s) of miR-1<em>2</em>5b in psoriasis pathogenesis. In situ hybridization results showed that the major cell type responsible for decreased miR-1<em>2</em>5b levels in psoriasis lesions was the <em>keratinocyte</em>. Overexpression of miR-1<em>2</em>5b in primary human <em>keratinocytes</em> suppressed proliferation and induced the expression of several known differentiation markers. Conversely, inhibition of endogenous miR-1<em>2</em>5b promoted cell proliferation and delayed differentiation. Fibroblast <em>growth</em> <em>factor</em> receptor <em>2</em> (FGFR<em>2</em>) was identified as one of the direct targets for suppression by miR-1<em>2</em>5b by luciferase reporter assay. The expression of miR-1<em>2</em>5b and FGFR<em>2</em> was inversely correlated in both transfected <em>keratinocytes</em> and in psoriatic skin. Knocking down FGFR<em>2</em> expression by siRNA suppressed <em>keratinocyte</em> proliferation, but did not enhance differentiation. Altogether, our results demonstrate a role for miR-1<em>2</em>5b in the regulation of <em>keratinocyte</em> proliferation and differentiation, partially through the regulation of FGFR<em>2</em>. Loss of miR-1<em>2</em>5b in psoriasis skin may contribute to hyperproliferation and aberrant differentiation of <em>keratinocytes</em>.

EGF family <em>growth</em> <em>factors</em>, including transforming <em>growth</em> <em>factor</em>-alpha (TGFalpha), amphiregulin (AR), and heparin-binding EGF (HB-EGF), are invariably expressed as transmembrane precursors that are cleaved at one or more sites in the extracellular domain to release soluble <em>growth</em> <em>factor</em>. Considerable attention has focused on the identification of proteases responsible for these processing events. We previously implicated tumor necrosis <em>factor</em>-alpha converting enzyme (TACE/ADAM17) in the generation of soluble TGFalpha from its transmembrane precursor, proTGFalpha. Here, we review our findings that primary <em>keratinocytes</em> from Tace(deltaZn/deltaZn) mice, which express a nonfunctional TACE, released dramatically lower levels of soluble TGFalpha compared to their normal counterparts, even though TGFalpha mRNA and cell-associated protein levels were similar in the two cell populations. Restoration of TACE activity in Tace(deltaZn/deltaZn) cells increased shedding of TGFalpha species, including the mature, 6-kDa protein. Further, exogenous TACE enzyme accurately cleaved the N-terminal processing site of proTGFalpha in cell lysates, as well as both physiologic sites of a soluble proTGFalpha ectodomain. TACE also accurately cleaved peptide substrates corresponding to the processing sites of several additional EGF family members, and restoration of TACE activity enhanced the shedding of soluble AR and HB-EGF proteins from Tace(deltaZn/deltaZn) cells. Finally, reduction of functional TACE gene dosage greatly exacerbated the open-eye defect of Egfr(wa-<em>2</em>/wa-<em>2</em>) newborns, which is regulated by redundant actions of several EGF family ligands. The implications of these results for the biology of the EGF family and TACE are discussed.

We sought to assess whether the effects of mesenchymal stromal cells (MSC) on lung inflammation and remodeling in experimental emphysema would differ according to MSC source and administration route. Emphysema was induced in C57BL/6 mice by intratracheal (IT) administration of porcine pancreatic elastase (0.1 UI) weekly for 1 month. After the last elastase instillation, saline or MSCs (1×105), isolated from either mouse bone marrow (BM), adipose tissue (AD) or lung tissue (L), were administered intravenously (IV) or IT. After 1 week, mice were euthanized. Regardless of administration route, MSCs from each source yielded: 1) decreased mean linear intercept, neutrophil infiltration, and cell apoptosis; <em>2</em>) increased elastic fiber content; 3) reduced alveolar epithelial and endothelial cell damage; and 4) decreased <em>keratinocyte</em>-derived chemokine (KC, a mouse analog of interleukin-8) and transforming <em>growth</em> <em>factor</em>-β levels in lung tissue. In contrast with IV, IT MSC administration further reduced alveolar hyperinflation (BM-MSC) and collagen fiber content (BM-MSC and L-MSC). Intravenous administration of BM- and AD-MSCs reduced the number of M1 macrophages and pulmonary hypertension on echocardiography, while increasing vascular endothelial <em>growth</em> <em>factor</em>. Only BM-MSCs (IV>> IT) increased the number of M<em>2</em> macrophages. In conclusion, different MSC sources and administration routes variably reduced elastase-induced lung damage, but IV administration of BM-MSCs resulted in better cardiovascular function and change of the macrophage phenotype from M1 to M<em>2</em>.

Although neutrophils are strongly implicated in eliminating pathogens, excessive recruitment may cause tissue damage. Therefore, reducing cell influx during an inflammatory process may be a potential target for treating inflammatory bowel diseases (IBD). As CXCR<em>2</em> is involved in neutrophil migration, this study aimed to evaluate whether the systemic therapeutic treatment with selective CXCR<em>2</em> antagonist SB<em>2</em><em>2</em>500<em>2</em> ameliorates experimental colitis, which was induced in mice by <em>2</em>,4,6-trinitrobenzene sulfonic acid (TNBS). After colitis establishment (<em>2</em>4 h), mice were treated with SB<em>2</em><em>2</em>500<em>2</em>. At later time-points, up to 7<em>2</em> h, mice were monitored for body weight loss and overall mortality. At the time of sacrifice, colonic tissues were scored for macro- and microscopic damage, and cytokine levels, myeloperoxidase (MPO) activity, and protein expression were analyzed. TNBS administration induced macro- and microscopic damage in colon tissue, leading in most cases to animal death. Curative treatment with SB<em>2</em><em>2</em>500<em>2</em> significantly reduced all of the parameters analyzed, leading to an improvement of inflammatory signs. SB<em>2</em><em>2</em>500<em>2</em> reduced neutrophil influx, MPO activity, IL-1beta, MIP-<em>2</em>, and <em>keratinocyte</em>-derived chemokine (KC) levels and the expression of vascular endothelial <em>growth</em> <em>factor</em>, inducible NO synthase, and cyclooxygenase-<em>2</em> proteins into the colon tissue. Levels of IL-4 and IL-10 were increased significantly in the colons of animals treated with SB<em>2</em><em>2</em>500<em>2</em>. Additionally, curative treatment with mouse anti-KC significantly reduced MPO activity and colonic damage. These results taken together demonstrate that a selective blockade of CXCR<em>2</em> consistently reduced TNBS-induced colitis, suggesting that the use of SB<em>2</em><em>2</em>500<em>2</em> is a potential therapeutic approach for the treatment of IBD and other related inflammatory disorders.

Collagenase-3 (MMP-13) is a human matrix metalloproteinase specifically expressed by transformed squamous epithelial cells, i.e. squamous cell carcinoma (SCC) cells in culture and in vivo. Here, we have elucidated the signaling pathways regulating MMP-13 expression in transformed human epidermal <em>keratinocytes</em>, i.e. ras-transformed HaCaT cell line A-5 and cutaneous SCC cell line (UT-SCC-7). Treatment with tumor necrosis <em>factor</em>-(alpha) (TNF-(alpha) resulted in activation of extracellular signal-regulated kinase (ERK)1,<em>2</em>, Jun N-terminal kinase and p38 mitogen-activated protein kinase (MAPK) in both cell lines. In addition, transforming <em>growth</em> <em>factor</em>-(beta) (TGF-(beta) activated p38 MAPK in both cell lines, and ERK<em>2</em> in A-5 cells. Selective inhibition of p38 activity with SB <em>2</em>03580 abolished the enhancement of MMP-13, as well as collagenase-1 (MMP-1) and 9<em>2</em>-kDa gelatinase (MMP-9) expression by TNF-(alpha) and TGF-(beta). Blocking the ERK1, <em>2</em> pathway by PD 98059 had no effect on the induction of MMP-13 expression by TNF-(alpha) or TGF-(beta), but potently suppressed MMP-1 and MMP-9 production. Inhibition of p38 activity by SB <em>2</em>03580 also suppressed collagenolytic activity produced by both cell lines and inhibited invasion of TNF-(alpha) or TGF-(beta) stimulated A-5 cells through type I collagen and reconstituted basement membrane (Matrigel). These results show that activation of p38 MAPK pathway plays a crucial role in the invasive phenotype of transformed squamous epithelial cells, suggesting p38 MAPK as a target to specifically inhibit their invasion.

BACKGROUND

Systemic sclerosis (SSc) is characterized by widespread microangiopathy, fibrosis, and autoimmunity. Despite the lack of angiogenesis, the expression of vascular endothelial growth factor A (VEGF) was shown to be upregulated in SSc skin and circulation; however, previous studies did not distinguish between proangiogenic VEGF(165) and antiangiogenic VEGF(165)b isoforms, which are generated by alternative splicing in the terminal exon of VEGF pre-RNA.

OBJECTIVE

We investigated whether VEGF isoform expression could be altered in skin and circulation of patients with SSc.

RESULTS

Here, we show that the endogenous antiangiogenic VEGF(165)b splice variant is selectively overexpressed at both the mRNA and protein levels in SSc skin. Elevated VEGF(165)b expression correlated with increased expression of profibrotic transforming growth factor-β1 and serine/arginine protein 55 splicing factor in keratinocytes, fibroblasts, endothelial cells, and perivascular inflammatory cells. Circulating levels of VEGF(165)b were significantly higher in patients with SSc than in control subjects. Microvascular endothelial cells (MVECs) isolated from SSc skin expressed and released higher levels of VEGF(165)b than healthy MVECs. Transforming growth factor-β1 upregulated the expression of VEGF(165)b and serine/arginine protein 55 in both SSc and healthy MVECs. In SSc MVECs, VEGF receptor-2 was overexpressed, but its phosphorylation was impaired. Recombinant VEGF(165)b and SSc-MVEC-conditioned medium inhibited VEGF(165)-mediated VEGF receptor-2 phosphorylation and capillary morphogenesis in healthy MVECs. The addition of anti-VEGF(165)b blocking antibodies abrogated the antiangiogenic effect of SSc-MVEC-conditioned medium. Capillary morphogenesis was severely impaired in SSc MVECs and could be ameliorated by treatment with recombinant VEGF(165) and anti-VEGF(165)b blocking antibodies.

CONCLUSIONS

In SSc, a switch from proangiogenic to antiangiogenic VEGF isoforms may have a crucial role in the insufficient angiogenic response to chronic ischemia.

During the wound healing process lysis of basement membranes precedes <em>keratinocyte</em> migration into the wound bed. We studied, in vitro, whether this degradation of basement membranes could be regulated by transforming <em>growth</em> <em>factor</em>-beta 1 (TGF-beta 1), which is known to accelerate wound healing in vivo. Transforming <em>growth</em> <em>factor</em>-beta 1 was found to increase the expression of both 9<em>2</em>- and 7<em>2</em>-kDa type IV collagenases (gelatinases) in cultured human mucosal and dermal <em>keratinocytes</em>. The 9<em>2</em>-kDa enzyme predominated in both unstimulated and stimulated cultures. The 9<em>2</em>-kDa form was stimulated over 5-fold, and the other form by a <em>factor</em> of <em>2</em>-3. This increase in the synthesis of type IV collagenases was associated with a marked increase in the mRNA levels of these enzymes as well. The induction of the 9<em>2</em>-kDa enzyme was similar in culture medium containing either 0.15 or 1.<em>2</em> mM calcium chloride. Rat mucosal <em>keratinocytes</em> secreted only 9<em>2</em>-kDa type IV collagenase, the secretion of which was not regulated by TGF-beta 1. Also, TGF-beta 1 did not cause any significant induction (maximum about 1.<em>2</em>-fold) of either type IV collagenase in human gingival fibroblasts. The induction levels of both collagenases in human <em>keratinocytes</em> were independent of the type of the extracellular matrix the cells were grown on. However, the basement membrane matrix (Matrigel) activated about half of the 9<em>2</em>-kDa type to its 84-kDa active form. The data suggest that TGF-beta 1 has a specific function in up-regulating the expression of type IV collagenases in human <em>keratinocytes</em>, offering a possible explanation of how <em>keratinocytes</em> detach from basement membranes prior to the migration over the wound bed.

BACKGROUND: Breast pain and tenderness affects 70% of women at some time. These symptoms have been attributed to stretching of the nerves with increase in breast size, but tissue mechanisms are poorly understood. METHODS: Eighteen patients (n = 1<em>2</em> breast reduction and n = 6 breast reconstruction) were recruited and assessed for breast pain by clinical questionnaire. Breast skin biopsies from each patient were examined using immunohistological methods with specific antibodies to the capsaicin receptor TRPV1, related vanilloid thermoreceptors TRPV3 and TRPV4, and nerve <em>growth</em> <em>factor</em> (NGF). RESULTS: TRPV1-positive intra-epidermal nerve fibres were significantly increased in patients with breast pain and tenderness (TRPV1 fibres / mm epidermis, median [range] - no pain group, n = 8, 0.69 [0-1.<em>2</em>7]; pain group, n = 10, <em>2</em>.15 [0.77-4.38]; p = 0.0009). Nerve <em>Growth</em> <em>Factor</em>, which up-regulates TRPV1 and induces nerve sprouting, was present basal <em>keratinocytes</em>: some breast pain specimens also showed NGF staining in supra-basal <em>keratinocytes</em>. TRPV4-immunoreactive fibres were present in sub-epidermis but not significantly changed in painful breast tissue. Both TRPV3 and TRPV4 were significantly increased in <em>keratinocytes</em> in breast pain tissues; TRPV3, median [range] - no pain group, n = 6, 0.75 [0-<em>2</em>]; pain group, n = 11, <em>2</em> 1<em>2</em>3, p = 0.008; TRPV4, median [range] - no pain group, n = 6, [0-1]; pain group, n = 11, 1 [0.5-<em>2</em>], p = 0.014). CONCLUSION: Increased TRPV1 intra-epidermal nerve fibres could represent collateral sprouts, or re-innervation following nerve stretch and damage by polymodal nociceptors. Selective TRPV1-blockers may provide new therapy in breast pain. The role of TRPV3 and TRPV4 changes in <em>keratinocytes</em> deserve further study.

Hypoxia inducible <em>factor</em>-1 (HIF-1) is a master regulatory transcription <em>factor</em> controlling multiple cell-autonomous and non-cell-autonomous processes, such as metabolism, angiogenesis, matrix invasion, and cancer metastasis. Here we used a new line of transgenic mice with constitutive gain of HIF-1 function in basal <em>keratinocytes</em> and demonstrated a signaling pathway from HIF-1 to nuclear <em>factor</em> kappa B (NFkappaB) activation to enhanced epithelial chemokine and cytokine elaboration. This pathway was responsible for a phenotypically silent accumulation of stromal inflammatory cells and a marked inflammatory hypersensitivity to a single 1<em>2</em>-O-tetradecanoylphorbol-13-acetate (TPA) challenge. HIF-1-induced NFkappaB activation was composed of <em>2</em> elements, IkappaB hyperphosphorylation and phosphorylation of Ser<em>2</em>76 on p65, enhancing p65 nuclear localization and transcriptional activity, respectively. NFkappaB transcriptional targets macrophage inflammatory protein-<em>2</em> (MIP-<em>2</em>/CXCL<em>2</em>/3), <em>keratinocyte</em> chemokine (KC/CXCL1), and tumor necrosis <em>factor</em> [alfa] (TNFalpha) were constitutively up-regulated and further increased after TPA challenge both in cultured <em>keratinocytes</em> and in transgenic mice. Whole animal KC, MIP-<em>2</em>, or TNFalpha immunodepletion each abrogated TPA-induced inflammation, whereas blockade of either VEGF or placenta <em>growth</em> <em>factor</em> (PlGF) signaling did not affect transgenic inflammatory hyper-responsiveness. Thus, epithelial HIF-1 gain of function remodels the local environment by cell-autonomous NFkappaB-mediated chemokine and cytokine secretion, which may be another mechanism by which HIF-1 facilitates either inflammatory diseases or malignant progression.

Hyaluronan is an abundant and rapidly turned over matrix molecule between the vital cell layers of the epidermis. In this study, epidermal <em>growth</em> <em>factor</em> (EGF) induced a coat of hyaluronan and a 3-5-fold increase in its rate of synthesis in a rat epidermal <em>keratinocyte</em> cell line that has retained its ability for differentiation. EGF also increased hyaluronan in perinuclear vesicles, suggesting concurrent enhancement in its endocytosis. Cell-associated hyaluronan was most abundant in elongated cells that were stimulated to migrate by EGF, as determined in vitro in a wound healing assay. Large fluctuations in the pool size of UDP-N-acetylglucosamine, the metabolic precursor of hyaluronan, correlated with medium glucose concentrations but not with EGF. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed no increase in hyaluronan synthases 1 and 3 (Has1 and Has3), whereas Has<em>2</em> mRNA increased <em>2</em>-3-fold in less than <em>2</em> h following the introduction of EGF, as estimated by quantitative RT-PCR with a truncated Has<em>2</em> mRNA internal standard. The average level of Has<em>2</em> mRNA increased from approximately 6 copies/cell in cultures before change of fresh medium, up to approximately 54 copies/cell after 6 h in EGF-containing medium. A control medium with 10% serum caused a maximum level of approximately <em>2</em>1 copies/cell at 6 h. The change in the Has<em>2</em> mRNA levels and the stimulation of hyaluronan synthesis followed a similar temporal pattern, reaching a maximum level at 6 h and declining toward <em>2</em>4 h, a finding in line with a predominantly Has<em>2</em>-dependent hyaluronan synthesis and its transcriptional regulation.

The vanilloid receptor-1 (VR1, or transient receptor potential vanilloid-1 receptor, TRPV1) is activated by capsaicin, the key ingredient of hot peppers. TRPV1 was originally described on sensory neurons as a central integrator of various nociceptive stimuli. However, several human skin cell populations are also now recognized to express TRPV1, but with unknown function. Exploiting the human hair follicle (HF) as a prototypic epithelial-mesenchymal interaction system, we have characterized the HF expression of TRPV1 in situ and have examined TRPV1 signaling in organ-cultured human scalp HF and outer root sheath (ORS) <em>keratinocytes</em> in vitro. TRPV1 immunoreactivity was confined to distinct epithelial compartments of the human HF, mainly to the ORS and hair matrix. In organ culture, TRPV1 activation by capsaicin resulted in a dose-dependent and TRPV1-specific inhibition of hair shaft elongation, suppression of proliferation, induction of apoptosis, premature HF regression (catagen), and up-regulation of intrafollicular transforming <em>growth</em> <em>factor</em>-beta(<em>2</em>). Cultured human ORS <em>keratinocytes</em> also expressed functional TRPV1, whose stimulation inhibited proliferation, induced apoptosis, elevated intracellular calcium concentration, up-regulated known endogenous hair <em>growth</em> inhibitors (interleukin-1beta, transforming <em>growth</em> <em>factor</em>-beta(<em>2</em>)), and down-regulated known hair <em>growth</em> promoters (hepatocyte <em>growth</em> <em>factor</em>, insulin-like <em>growth</em> <em>factor</em>-I, stem cell <em>factor</em>). These findings strongly support TRPV1 as a significant novel player in human hair <em>growth</em> control, underscore the physiological importance of TRPV1 in human skin beyond nociception, and identify TRPV1 as a promising, novel target for pharmacological manipulations of epithelial <em>growth</em> disorders.

<em>Keratinocytes</em> and dermal endothelial cells, excluding leukocytes that infiltrate wounds, are the main source of soluble <em>factors</em> regulating healing of skin ulcers. We used immunohistochemistry to analyze the expression of various chemotactic and <em>growth</em> <em>factors</em> and their receptors in the margin of diabetic foot ulcers and in normal nondiabetic foot skin. Our study found significantly elevated expression of transforming <em>growth</em> <em>factor</em>-beta1 (TGF-beta1) and type I TGF-beta receptors (TGFbetaR1), granulocyte macrophage colony-stimulating <em>factor</em> (GM-CSF), and epidermal <em>growth</em> <em>factor</em> (EGF) in <em>keratinocytes</em> in the ulcer margin (p < 0.05). Significantly increased expression of monocyte chemotactic protein-1, GM-CSF, CXCR1, and TGFbetaRI and decreased expression of interleukin (IL)-10, IL-15, and TGF-beta1 were observed in ulcer dermal endothelial cells (p < 0.05). There was a lack of up-regulation of IL-8, CCR<em>2</em>A, IL-10 receptor, GM-CSF receptor, platelet-derived <em>growth</em> <em>factors</em> and their receptors, vascular endothelial <em>growth</em> <em>factor</em> and its type II receptor, EGF receptor, insulin-like <em>growth</em> <em>factor</em>-1, and nitric oxide synthase-<em>2</em> in both KCs and endothelial cells in the ulcer. Finally, there was a lack of up-regulation of IL-10 and IL-15 in <em>keratinocytes</em> and of EGF, basic fibroblast <em>growth</em> <em>factor</em>, and nitric oxide synthase-3 in endothelial cells in the ulcer margins. The enhanced expression of some <em>factors</em> responsible for KC behavior could suggest an unimpaired capacity of <em>keratinocytes</em> to reepithelialize the margin of diabetic foot ulcers. However, lack of up-regulation of some angiogenic and leukocyte chemotactic <em>factors</em>, associated with the reduced influx of immune cells, may account for a poor formation of granulation tissue and chronicity of ulcer epithelialization.

Keloids are locally aggressive scars that typically invade into healthy surrounding skin and cause both physical and psychosocial distress to the patient. These pathological scars occur following minimal skin trauma after a variety of causes including burns and trauma. Although the pathogenesis of keloid disease is not well understood, it is considered to be the end product of an abnormal healing process. The aim of this review was to investigate the molecular and cellular pathobiology of keloid disease in relation to the normal wound healing process. The molecular aberrances in keloids that correlate with the molecular mechanisms in normal wound healing can be categorized into three groups: (1) extracellular matrix proteins and their degradation, (<em>2</em>) cytokines and <em>growth</em> <em>factors</em>, and (3) apoptotic pathways. With respect to cellular involvements, fibroblasts are the most well-studied cell population. However, it is unclear whether the fibroblast is the causative cell; they are modulated by other cell populations in wound repair, such as <em>keratinocytes</em> and macrophages. This review presents a detailed account of individual phases of the healing process and how they may potentially be implicated in aberrant raised scar formation, which may help in clarifying the mechanisms involved in keloid disease pathogenesis.

The Forkhead family of transcription <em>factors</em> participates in the induction of death-related genes. In NMuMG and 4T1 mammary epithelial cells, transforming <em>growth</em> <em>factor</em> beta (TGF beta) induced phosphorylation and cytoplasmic retention of the Forkhead <em>factor</em> FKHRL1, while reducing FHKRL1-dependent transcriptional activity. TGF beta-induced FKHRL1 phosphorylation and nuclear exclusion were inhibited by LY<em>2</em>9400<em>2</em>, an inhibitor of phosphatidylinositol-3 kinase. A triple mutant of FKHRL1, in which all three Akt phosphorylation sites have been mutated (TM-FKHRL1), did not translocate to the cytoplasm in response to TGF beta. In HaCaT <em>keratinocytes</em>, expression of dominant-negative Akt prevented TGF beta-induced 1) reduction of Forkhead-dependent transcription, <em>2</em>) FKHRL1 phosphorylation, and 3) nuclear exclusion of FKRHL1. Forced expression of either wild-type (WT) or TM-FKHRL1, but not a FKHRL1 mutant with deletion of the transactivation domain, resulted in NMuMG mammary cell apoptosis. Evidence of nuclear fragmentation colocalized to cells with expression of WT- or TM-FKHRL1. The apoptotic effect of WT-FKHRL1 but not TM-FKHRL1 was prevented by exogenous TGF beta. Serum starvation-induced apoptosis was also inhibited by TGF beta in NMuMG and HaCaT cells. Finally, dominant-negative Akt abrogated the antiapoptotic effect of TGF beta. Taken together, these data suggest that TGF beta may play a role in epithelial cell survival via Akt-dependent regulation of FKHRL1.

OBJECTIVE

To examine the clinical progression and innate immune responses during Pseudomonas aeruginosa (PA) keratitis in cathelicidin-deficient (KO) mice.

METHODS

PA (ATCC 19660) keratitis was induced in KO mice and wild-type (WT) littermates generated on a 1<em>2</em>9/SVJ background. Clinical score and histopathology were used to monitor the progression of infection at postinfection (PI) days 1, 3, 7, 14, and <em>2</em>1. Mouse corneas were harvested for viable bacteria quantitation, and myeloperoxidase (MPO) assays were performed to determine the number of infiltrating neutrophils. ELISA was used to quantitate interleukin (IL)-1beta, IL-6, macrophage inflammatory peptide (MIP)-<em>2</em>, <em>keratinocyte</em>-derived chemokine (KC), tumor necrosis <em>factor</em> (TNF)-alpha, and vascular endothelial <em>growth</em> <em>factor</em> (VEGF) levels in the corneas.

RESULTS

WT mice were resistant (cornea healed), whereas KO mice showed increased susceptibility (corneas failed to recover by <em>2</em>1 days or perforated) to PA infection. Clinical scores were significantly elevated in the infected corneas of KO mice versus WT mice at 7, 14, and <em>2</em>1 days PI. Absence of cathelicidin resulted in significantly delayed clearance of PA in the cornea and an increased number of infiltrating neutrophils at 1, 3, 7, and 14 days PI. KO mice also exhibited differential expression of protein levels for IL-1beta, IL-6, MIP-<em>2</em>, KC, TNF-alpha, and VEGF up to day <em>2</em>1 PI compared with the WT mice.

CONCLUSIONS

Cathelicidin-deficient mice showed considerable susceptibility to PA keratitis. The present study demonstrates direct in vivo evidence that endogenous expression of cathelicidin provides defense against corneal PA infection indicating its importance in host innate immunity at the ocular surface.

Purified type beta transforming <em>growth</em> <em>factor</em> from human platelets (TGF beta) radioiodinated with 1<em>2</em>5I-labeled Bolton and Hunter reagent was found to bind to a variety of cultured cells of both epithelial and mesenchymal origin, including normal human fibroblasts and <em>keratinocytes</em>. TGF beta binding sites have also been found on three mouse embryo-derived fibroblast-like cell lines with lower levels of TGF beta binding on the chemically transformed derivatives of these cell lines. A variety of human tumor cell lines was shown to have an inverse correlation between their level of TGF beta binding and their ability to form colonies in soft agar. The mouse embryo-derived AKR-<em>2</em>B (clone 84A) cells reached maximal binding of 1<em>2</em>5I-labeled TGF beta after <em>2</em> hr at <em>2</em><em>2</em> degrees C. Scatchard analysis of the equilibrium binding of TGF beta to AKR-<em>2</em>B (clone 84A) cells gives a Kd of 33 pM with approximately equal to 10,500 binding sites per cell. This Kd for TGF beta binding to AKR-<em>2</em>B (clone 84A) cells agreed well with the ED50 of 40 pM for stimulation of colony formation of these cells by TGF beta. The TGF beta binding sites on the AKR-<em>2</em>B cells were shown to be specific for TGF beta with no significant competition with epidermal <em>growth</em> <em>factor</em>, fibroblast <em>growth</em> <em>factor</em>, or insulin and only a small level of competition with high concentrations of platelet-derived <em>growth</em> <em>factor</em>. Partially purified preparations with TGF beta-like activity from mouse embryos and medium conditioned by mouse embryo-derived cells competed effectively for binding to the TGF beta receptor.