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The ZFP36 family of zinc finger proteins, including ZFP36, ZFP36L1, and ZFP36L2, regulates the production of growth factors and cytokines via destabilization of the respective mRNAs. We could recently demonstrate that in cultured keratinocytes, expression of the ZFP36, ZFP36L1, and ZFP36L2 genes is induced by growth factors and cytokines and that ZFP36L1 is a potent regulator of keratinocyte VEGF production. We now further analyzed the localization and function of ZFP36 proteins in the skin, specifically in epidermal keratinocytes. We found that in human epidermis, the ZFP36 protein could be detected in basal and suprabasal keratinocytes, whereas ZFP36L1 and ZFP36L2 were expressed mainly in the basal layer, indicating different and non-redundant functions of the three proteins in the epidermis. Consistently, upon inhibition of ZFP36 or ZFP36L1 expression using specific siRNAs, there was no major effect on expression of the respective other gene. In addition, we demonstrate that both ZFP36 and ZFP36L1 influence keratinocyte cell cycle, differentiation, and apoptosis in a distinct manner. Finally, we show that similarly as ZFP36L1, ZFP36 is a potent regulator of keratinocyte VEGF production. Thus, it is likely that both proteins regulate angiogenesis via paracrine mechanisms. Taken together, our results suggest that ZFP36 proteins might control reepithelialization and angiogenesis in the skin in a multimodal manner., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Keratinocyte-derived growth factors and cytokines play an important role in epidermal homeostasis and particularly in cutaneous wound repair. Thus, we analyzed a potential role of the ZFP36/tristetraprolin family of zinc finger proteins, which are targets of these factors, but also regulate their production, in keratinocytes. We show that expression of ZFP36, ZFP36L1, and ZFP36L2 is induced by a broad variety of growth factors and cytokines, and by scratch wounding. Since ZFP36L1 is a modulator of vascular endothelium growth factor (VEGF) mRNA stability, we subsequently used siRNA technology to inhibit ZFP36L1 gene expression. Notably, this treatment resulted in prolonged maintenance of elevated VEGF levels in HaCaT keratinocytes upon epidermal growth factor stimulation of these cells. Taken together, our results suggest an important role of ZFP36L1 in wound healing.

We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-alpha-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients., (2009 Elsevier Inc. All rights reserved.)

Receptor interacting protein 4 (RIP4) is an important regulator of epidermal morphogenesis during embryonic development. We could previously show that expression of the rip4 gene is strongly downregulated in cutaneous wound repair, which might be initiated by a broad variety of growth factors and cytokines. Here, we demonstrate that in keratinocytes, rip4 expression is controlled by a multitude of different signal transduction pathways, such as the p38 mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B (NF-kappaB) cascade, in a unique and specific manner. Furthermore, we show that the steroid dexamethasone abolishes the physiological rip4 downregulation after injury and might thus contribute to the phenotype of reduced and delayed wound reepithelialization seen in glucocorticoid-treated patients. As a whole, our data indicate that rip4 expression is regulated in a complex manner, which might have therapeutic implications.

Receptor-interacting proteins (RIPs) are important regulators of cell proliferation and differentiation. As RIP4 is a crucial modulator of epidermal differentiation, we analyzed the expression of different rip genes in healing skin wounds. Rip4 expression was strongly downregulated in keratinocytes of the hyperproliferative epithelium at the wound edge early after injury and only returned to basal levels after completion of wound repair. Rip3 expression was strongly induced as early as 1 day after wounding. In contrast, rip and rip2 expression remained unaltered. To determine the factors that regulate rip4 gene expression in keratinocytes, human HaCaT keratinocytes were used as a model system. We found that scratch wounding as well as treatment with whole serum, phorbol esters, the growth/differentiation factors epidermal growth factor, transforming growth factor-beta, and activin A, or the proinflammatory cytokines tumor necrosis factor-alpha and IL-1beta strongly suppressed rip4 expression in these cells. In contrast, the steroid dexamethasone and all-trans retinoic acid slightly stimulated rip4 expression. Suppression of rip4 expression in keratinocytes using small interfering RNA technology reduced the activation of NF-kappaB, and enhanced the expression of epidermal differentiation markers in these cells. These data suggest important and unique functions of different RIP proteins in keratinocytes of normal and wounded skin.

Hepatocyte growth-factor (HGF) is a potent, widely produced, pleiotropic mediator of mesenchymal-epithelial interaction. In a study of changes in gene expression initiated by HGF in Balb/MK keratinocytes, we observed the induction of Neu-differentiation factor (NDF) mRNA (also known as heregulin, or HRG). Further characterization of the regulation of NDF expression in Balb/MK keratinocytes revealed potent induction by keratinocyte growth factor (KGF) and epidermal growth factor (EGF), but not by HGF/NK2, an alternative HGF isoform with motogenic but not mitogenic or morphogenic activities. Sustained treatment (8 h) of Balb/MK cells with KGF stimulated secretion of mature NDF protein into the culture medium, and Balb/MK cells treated with purified recombinant NDF protein showed increased DNA synthesis. We also found evidence of NDF induction in two models of tissue repair in mice: in full-thickness skin wounds, following locally increased KGF production, and in kidney after partial hepatectomy, following elevation of circulating HGF levels. These results reveal that mesenchymally-derived HGF and KGF can activate autocrine NDF signaling in their epithelial targets, and suggest that this mechanism contributes to the coordination of stages of wound repair, and possibly development, where these growth factors act in concert to direct epithelial proliferation, morphogenesis and differentiation.Oncogene (2000) 19, 640–648. [ABSTRACT FROM AUTHOR]

Recently we demonstrated a strong induction of activin expression after skin injury, suggesting a function of this transforming growth factor-β family member in wound repair. To test this possibility, we generated transgenic mice that overexpress the activin βA chain in the epidermis under the control of a keratin 14 promoter. The transgenic mice were significantly smaller than control litter mates, and they had smaller ears and shorter tails. In their skin, the fatty tissue was replaced by connective tissue and a severe thickening of the epidermis was found. The spinous cell layer was significantly increased, and the epidermal architecture was highly disorganized. These histological abnormalities seem to result from increased proliferation of the basal keratinocytes and abnormalities in the program of keratinocyte differentiation. After skin injury, a significant enhancement of granulation tissue formation was detected in the activin­overexpressing mice, possibly as a result of premature induction of fibronectin and tenascin-C expression. These data reveal novel activities of activin in the regulation of keratinocyte proliferation and differentiation as well as in dermal fibrosis and cutaneous wound repair. [ABSTRACT FROM AUTHOR]

Keratinocyte growth factor is a potent and specific mitogen for different types of epithelial cells, including keratinocytes of the skin. Furthermore, it has been implicated in morphogenetic processes of several organs. To further define the mechanisms of KGF action in the skin, we attempted to identify genes which are regulated by KGF in keratinocytes. Using the differential display RT – PCR technology, a gene was identified which was strongly induced in these cells by treatment with KGF but not with serum growth factors or pro-inflammatory cytokines. Molecular cloning of the full-length cDNA revealed a strong homology of the corresponding gene product with a bovine non-selenium glutathione peroxidase. Upon transfection of COS cells with the full-length cDNA, a 27 kD cytoplasmic protein was obtained which had the expected size of glutathione peroxidase. Expression of the novel gene was detected in normal human skin and at particularly high levels in psoriatic skin, indicating a possible in vivo function of the protein in this tissue. Identification of a peroxidase as a KGF-regulated gene suggests that prevention from oxygen toxicity is a novel and specific mechanism of KGF action. [ABSTRACT FROM AUTHOR]

Transforming growth factor β1 (TGF-β1) is an important modulator of skin morphogenesis and cutaneous wound repair. To gain insight into the mechanisms of TGF-β1 action in the skin, we used the differential display RT-PCR technique to identify genes that are regulated by this factor in cultured human keratinocytes. We obtained several partial cDNA clones. One of them was identical to the 3'-end of p11, the small and regulatory subunit of the calpactin I complex [(annexin II)2(p11)2]. RNase protection and northern blot analysis revealed specific regulation of expression of both subunits of this heterotetrameric protein (p11 and annexin II) by TGF-β1 as well as by other growth factors, although the time course and degree of induction or suppression were different for each gene. Furthermore, we analyzed p11 and annexin II expression in normal and wounded skin. Both p11 and annexin II mRNAs were found in the dermal and epidermal compartments of normal human skin. Immunohistochemical studies demonstrated the presence of p11 at equally high levels in all layers of normal epidermis and in the hyper-proliferative epithelium at the wound edge. By contrast, annex, in II expression was high in the basal layer of normal epidermis but low in the suprabasal layers and in the hyper-proliferative epithelium at the wound edge, suggesting a differentiation-specific regulation of this calpaetin I subunit. The differential expression and regulation of p11 and annexin II subunits in keratinocytes suggest the existence of different ratios of monomeric versus p11-complexed annexin II that might be associated with different cellular functions. [ABSTRACT FROM AUTHOR]