TGF-alpha regulates TLR expression and function on epidermal keratinocytes

The expression of TLRs on epithelial cells provides a first line of defense against invading pathogens. We investigated the regulated expression and function of TLR5 and TLR9 on human keratinocytes, because we found by immunohistochemistry that these TLRs are expressed in distinct layers of the epidermis. We found that TGF-alpha, a growth and differentiation factor that is present during wound healing and in psoriasis, increased the expression of both TLR5 and TLR9 on keratinocytes. In addition, TGF-alpha regulated the function of TLR5 and TLR9, because activation with their respective ligands enhanced the production of IL-8 and human beta-defensins. These findings provide evidence that TGF-alpha up-regulates TLR expression and function, augmenting host defense mechanisms at epithelial surfaces.     

MicroRNA‐181b negatively regulates the proliferation of human epidermal keratinocytes in psoriasis through targeting TLR4

Our study aims to explore the role of microRNA‐181b (miR‐181b) and TLR in the regulation of cell proliferation of human epidermal keratinocytes (HEKs) in psoriasis. Twenty‐eight patients diagnosed with psoriasis vulgaris were selected as a case group with their lesional and non‐lesional skin tissues collected. A control group consisted of 20 patients who underwent plastic surgery with their healthy skin tissues collected. Real‐time quantitative fluorescence polymerase chain reaction (RT‐qPCR), in situ hybridization and immunohistochemistry were used to detect the expressions of miR‐181b and TLR4 in HEKs of healthy skin, psoriatic lesional skin and non‐lesional skin respectively. The 3′ untranslated region (3′UTR) of TLR4 combined with miR‐181b was verified by a dual‐luciferase reporter assay. Western blotting and bromodeoxyuridine were applied for corresponding detection of TLR4 expression and cell mitosis. The expression of miR‐181b in HEKs of psoriatic lesional skin was less than healthy skin and psoriatic non‐lesional skin. In psoriatic lesional and non‐lesional skin, TLR4‐positive cell rates and the number of positive cells per square millimetre were higher than healthy skin. The dual‐luciferase reporter assay verified that miR‐181b targets TLR4. HEKs transfected with miR‐181b mimics had decreased expression of TLR4, along with the decrease of mitotic indexes and Brdu labelling indexes. However, HEKs transfected with miR‐181b inhibitors showed increased TLR4 expression, mitotic indexes and Brdu labelling indexes. HEKs transfected with both miR‐181b inhibitors and siTLR4 had decreased mitotic indexes and Brdu labelling indexes. These results indicate that miR‐181b can negatively regulate the proliferation of HEKs in psoriasis by targeting TLR4.     

Cholesterol inhibits MMP-9 expression in human epidermal keratinocytes and HaCaT cells

Cholesterol is a major component of skin lipids and acts as a regulator of vesicular trafficking and signal transduction. However, the function of cholesterol on matrix metalloproteinases (MMPs) expression of human skin is not fully understood. Here, we investigated the effects of cholesterol on MMP-9 expression in normal human keratinocytes (NHK) and HaCaT cells. Basal level of MMP-9 expression was decreased by cholesterol in NHK. On the other hand, MMP-9 expression was increased by the cholesterol depletion agent, methyl-beta-cyclodextrin (MbetaCD), while it was inhibited by cholesterol repletion in HaCaT cells. MbetaCD induced ERK and JNK phosphorylation were prevented by cholesterol repletion. The inhibition of ERK and JNK decreased MbetaCD-induced MMP-9 expression. Therefore, our results suggest that cholesterol regulates MMP-9 expression through ERK and JNK-dependent pathways.     

The tetraspanin CD9 associates with the integrin alpha6beta4 in cultured human epidermal keratinocytes and is involved in cell motility

Integrins are involved in several ways in keratinocyte physiology, including cell motility. CD9 is a member of the tetraspanin protein family which is found in association with other transmembrane proteins like the integrins. CD9 is expressed in the epidermal tissue, but this expression is not regulated by differentiation. The present work focuses on association of CD9 with the integrin alpha6beta4 in keratinocytes. In vivo, CD9 does not co-localize with alpha6beta4, and is not internalized with the integrin upon basal detachment with dispase. In vitro, CD9 is found partly in co-localization with alpha6beta4 and is internalized with the integrin after keratinocyte detachment with dispase. Using blocking antibodies in a phagokinetic tracks assay, we show that CD9, and to a lesser extent alpha6beta4, but not the tetraspanin CD82, promote motility of subconfluent keratinocytes on collagen I. Our observations also suggest that CD9 is involved in the formation of lamellipodia. We also report that the phorbol ester TPA has no effect on CD9 expression and association with alpha6beta4, but increases keratinocyte motility, possibly through modulation of integrin subunits expression, or through upregulation of collagenase-1 expression. Together, these results confirm that CD9 associates with alpha6beta4 in cultured keratinocytes, possibly in order to regulate the function of the integrin, and that CD9 is involved in keratinocyte motility on collagen. The data suggest that regulation of adhesion characteristics by CD9 in keratinocytes may play a role in epidermal repair.     

Deposition of laminin 5 by keratinocytes regulates integrin adhesion and signaling

Deposition of laminin 5 over exposed dermal collagen in epidermal wounds is an early event in repair of the basement membrane. We report that deposition of laminin 5 onto collagen switches adhesion and signaling from collagen-dependent to laminin 5-dependent. Ligation of laminin 5 by integrin alpha(6)beta(4) activates phosphoinositide 3-OH-kinase (PI3K) signaling. This activation allows for adhesion and spreading via integrin alpha(3)beta(1) on laminin 5 independent of RhoGTPase, a regulator of actin stress fibers. In contrast, adhesion and spreading on collagen via alpha(2)beta(1) is Rho-dependent and is inhibited by toxin B, a Rho inhibitor. Deposition of laminin 5 and ligation of alpha(6)beta(4) increases PI3K-dependent production of phosphoinositide di- and triphosphates, PI3K activity, and phosphorylation of downstream target protein c-Jun NH(2)-terminal kinase. Conversely, blocking laminin 5-deposition with brefeldin A, an inhibitor of vesicle transport, or with anti-laminin 5 monoclonal antibodies abolishes the PI3K-dependent spreading mediated by alpha(3)beta(1) and phosphorylation of c-Jun NH(2)-terminal kinase. Studies with keratinocytes lacking alpha(6)beta(4) or laminin 5 confirm that deposition of laminin 5 and ligation by alpha(6)beta(4) are required for PI3K-dependent spreading via alpha(3)beta(1). We suggest that deposition of laminin 5 onto the collagen substratum, as in wound repair, enables human foreskin keratinocytes to interact via alpha(6)beta(4) and to switch from a RhoGTPase-dependent adhesion on collagen to a PI3K-dependent adhesion and spreading mediated by integrin alpha(3)beta(1) on laminin 5.     

Gene expression profiling of human epidermal keratinocytes in simulated microgravity and recovery cultures

Simulated microgravity （SMG） bioreactors and DNA microarray technology are powerful tools to identify ＂space genes＂ that play key roles in cellular response to microgravity. We applied these biotechnology tools to investigate SMG and post-SMG recovery effects on human epidermal keratinocytes by exposing cells to SMG for 3, 4, 9, and 10 d using the high aspect ratio vessel bioreactor followed by recovery culturing for 15, 50, and 60 d in normal gravity. As a result, we identified 162 differentially expressed genes, 32 of which were ＂center genes＂ that were most consistently affected in the time course experiments. Eleven of the center genes were from the integrated stress response pathways and were coordinately downregulated. Another seven of the center genes, which are all metallothionein MT-Ⅰ and MT-Ⅱ isoforms, were coordinately up-regulated. In addition, HLA-G, a key gene in cellular immune response suppression, was found to be significantly upregulated during the recovery phase. Overall, more than 80% of the differentially expressed genes from the shorter exposures （≤4 d） recovered in 15 d; for longer （≥9 d） exposures, more than 50 d were needed to recover to the impact level of shorter exposures. The data indicated that shorter SMG exposure duration would lead to quicker and more complete recovery from the microgravity effect.     

MicroRNA-223 regulates FOXO1 expression and cell proliferation

In HCT116 colorectal cancer cells, HeLa cervical cancer cells and HuH-7 hepatoma cells, miR-223 is expressed at a low level. Through infection with lentivirus containing miR-223 precursor, miR-233 was overexpressed in all these cells. Interestingly, the expression levels of FOXO1 mRNA and protein, and phosphorylation levels became significantly lower than those of their control. FOXO1 was down-regulated mainly in the cytoplasm, while the nuclear FOXO1 level became relatively high compared to the cytoplasm. As the unphosphorylated active form of FOXO1 increased in the cells, cyclin D1/p21/p27 were up-regulated at either mRNA or protein level. Proliferation of the cells was also greatly inhibited when miR-223 was over-expressed. Therein, our data suggest that miR-223 regulates FOXO1 expression and cell proliferation.     

Retinoic acid potentiates TNF-alpha-induced ICAM-1 expression in normal human epidermal keratinocytes

ICAM-1 protein in keratinocytes is thought to contribute to cutaneous inflammatory reactions. Its induction depends-among others-on cytokines such as TNF-alpha, IFN-gamma, IL-1 or on retinoic acid (RA), a key regulator of epidermal homeostasis. We investigated the effect of treatments with TNF-alpha, RA or their combination on ICAM-1 expression on proliferative or differentiating keratinocytes over an 8 day culture period. Basal ICAM-1 levels were undetectable at low (30 microM) and standard (88 microM) Ca2+ and RA alone did not induce ICAM-1. However, at high Ca2+ (1500 microM), ICAM-1 levels were augmented in response to RA-treatment. TNF-alpha induced a transient ICAM-1 increase in NHK, which reached peak-levels 2-4 days post cytokine stimulus. RA potentiated the TNF-alpha-induced ICAM-1 response in all Ca2+-concentrations. This potentiating effect of RA was confirmed at the mRNA level. In summary, our results establish retinoic acid as an enhancer of TNF-alpha-induced ICAM-1 levels in NHK.     

Insulin-like growth factor-II regulates the 12-lipoxygenase gene expression and promotes cell proliferation in human keratinocytes via the extracellular regulatory kinase and phosphatidylinositol 3-kinase pathways

To study the relationship between insulin-like growth factor-II (IGF-II) and 12-lipoxygenase (12-LOX) that are upregulated in psoriasis, we monitored 12-lipoxygenase expression in the insulin-like growth factor-II treated human keratinocytes and explored the signaling pathways of 12-lipoxygenase expression. Insulin-like growth factor-II induced 12-lipoxygenase mRNA and protein levels in human keratinocytes through two major signal transduction pathways, namely, the extracellular signaling-regulated kinase (ERK)-mitogen activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) pathways. The IGF-II-induced upregulation of 12-lipoxygenase was attenuated by pretreating the cells with selective inhibitors or by overexpressing dominant-negative MEK. In addition, treatment of HaCaT cells with the 12-lipoxygenase metabolite 12 (S)-hydroxyeicosatetraenoic acid (12(S)-HETE) directly stimulated DNA synthesis and mitogenesis, and injection of insulin-like growth factor-II into the skin of hairless mice induced epidermal hyperplasia. These results suggest that insulin-like growth factor-II is involved in the pathogenesis of psoriasis as a paracrine inducer of 12-lipoxygenase.     

Substrate rigidity regulates human T cell activation and proliferation

Adoptive immunotherapy using cultured T cells holds promise for the treatment of cancer and infectious disease. Ligands immobilized on surfaces fabricated from hard materials such as polystyrene plastic are commonly employed for T cell culture. The mechanical properties of a culture surface can influence the adhesion, proliferation, and differentiation of stem cells and fibroblasts. We therefore explored the impact of culture substrate stiffness on the ex vivo activation and expansion of human T cells. We describe a simple system for the stimulation of the TCR/CD3 complex and the CD28 receptor using substrates with variable rigidity manufactured from poly(dimethylsiloxane), a biocompatible silicone elastomer. We show that softer (Young's Modulus [E] < 100 kPa) substrates stimulate an average 4-fold greater IL-2 production and ex vivo proliferation of human CD4(+) and CD8(+) T cells compared with stiffer substrates (E > 2 MPa). Mixed peripheral blood T cells cultured on the stiffer substrates also demonstrate a trend (nonsignificant) toward a greater proportion of CD62L(neg), effector-differentiated CD4(+) and CD8(+) T cells. Naive CD4(+) T cells expanded on softer substrates yield an average 3-fold greater proportion of IFN-γ-producing Th1-like cells. These results reveal that the rigidity of the substrate used to immobilize T cell stimulatory ligands is an important and previously unrecognized parameter influencing T cell activation, proliferation, and Th differentiation. Substrate rigidity should therefore be a consideration in the development of T cell culture systems as well as when interpreting results of T cell activation based upon solid-phase immobilization of TCR/CD3 and CD28 ligands.     

Deposition of laminin 5 in epidermal wounds regulates integrin signaling and adhesion

Adhesion of keratinocytes in a wound outgrowth to laminin 5 in the basement membrane via integrins alpha6beta4 and alpha3beta1 is distinct from adhesion to dermal collagen via alpha2beta1 or to fibronectin via alpha5beta1. Leading cells in the outgrowth are distinguished from following keratinocytes by deposition of laminin 5, failure to communicate via gap junctions and sensitivity to toxin B, an inhibitor of RhoGTPase. Laminin 5 deposited by leading keratinocytes onto dermal collagen dominates over dermal ligands and changes the cell signals required for adhesion from collagen-dependent to laminin-5-dependent. Thus, deposition of laminin 5 can instruct keratinocytes to switch from an activated phenotype to a quiescent and integrated epithelial phenotype.     

Novel protein in human epidermal keratinocytes: regulation of expression during differentiation.

Recently, two groups of cDNA clones have been isolated from human epidermal keratinocytes; the clones correspond to genes whose expression is stimulated by exposure of the cells to UV light or treatment with 4-nitroquinoline 1-oxide or 12-O-tetradecanoylphorbol 13-acetate (T. Kartasova and P. van de Putte, Mol. Cell. Biol. 8:2195-2203, 1988). The proteins predicted by the nucleotide sequence of both groups of cDNAs are small (8 to 10 kilodaltons), are exceptionally rich in proline, glutamine, and cysteine, and contain repeating elements with a common sequence, PK PEPC. These proteins were designated sprI and sprII (small, proline rich). Here we describe the characterization of the sprIa protein, which is encoded by one of the group 1 cDNAs. The expression of this protein during keratinocyte differentiation in vitro and the distribution of the sprIa protein in some human tissues was studied by using a specific rabbit antiserum directed against a synthetic polypeptide corresponding to the 30 amino acids of the C-terminal region of the sprIa gene product. The results indicate that the expression of the sprIa protein is stimulated during keratinocyte differentiation both in vitro and in vivo.     

Stanniocalcin-1 regulates re-epithelialization in human keratinocytes

Stanniocalcin-1 (STC1), a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam) formation, followed by cell migration. In this study, staurosporine (STS) treatment induced human keratinocyte (HaCaT) e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK), the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl) could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.     

JunD regulates lymphocyte proliferation and T helper cell cytokine expression

Transgenic mice broadly expressing JunD (Ubi-junD(m)) appear phenotypically normal, but have strongly reduced numbers of peripheral lymphocytes. JunD overexpression in lymphocytes does not protect from numerous apoptotic insults; however, transgenic T cells proliferate poorly and exhibit impaired activation due to reduced levels of IL-4, CD25 and CD69. Consistently, in the absence of JunD (junD(-/-)) T cells hyperproliferate following mitogen induction. Moreover, transgenic T helper (Th) 2 cells have decreased IL-4 and IL-10 expression, whereas junD(-/-) Th2 cells secrete higher amounts of both Th2 cytokines. Th1-polarized junD(-/-) CD4(+) T cells display enhanced IFN-gamma cytokine production associated with upregulated T-bet expression and downregulated expression of suppressor of cytokine signaling-1. These novel findings demonstrate a regulatory role of JunD in T lymphocyte proliferation and Th cell differentiation.     

Tissue-specific expression of keratin proteins in human esophageal and epidermal epithelium and their cultured keratinocytes

In contrast to the simplified keratin content of bovine, rabbit, and rat esophageal epithelium (composed mainly of a 57 and 46 or 51 kD keratin, depending on the animal species), human esophageal epithelium contained a quantitatively different array of keratin proteins, ranging in molecular weight from 37 to 61 kD. The pattern of keratin proteins from human esophageal epithelium differed qualitatively and quantitatively from that of human epidermis. Human esophageal epithelium lacked the 63, 65, and 67 kD keratins characteristic of human epidermis, consistent with the absence of a granular layer and an anucleate stratum corneum. Moreover, human esophageal epithelium contained a distinctive 61 kD keratin protein which was either not present or present in only small amounts in human epidermis and variable amounts of a 37 kD keratin. Whereas the 56, 59, and 67 kD keratins were the most abundant keratins in human epidermis, the 52, 57, and 61 kD keratins predominated in human esophageal epithelium. During in vitro cultivation, both human epidermal and esophageal keratinocytes produce colonies which are stratified, but the morphologic appearance of these cultured epithelia differs. Only cultured human epidermal keratinocytes contain keratohyalin granules in the outermost layers and a prominent 67 kD keratin on immunoprecipitation. Otherwise the keratin contents appear similar. In conclusion, human esophageal epithelium exhibited intertissue and interspecies differences in the pattern of keratin proteins. During in vitro cultivation, human esophageal keratinocytes retained some aspects of their distinctive program of differentiation.