Alteration of the TGF-beta/SMAD pathway in intrinsically and UV-induced skin aging

In an effort to characterize transforming growth factor (TGF-beta) signaling and to determine its association with the aging and photoaging processes, we directly compared the expressions of TGF-beta/SMAD in intrinsically aged and photoaged human skin in vivo. By using an RNase protection assay and by immunohistochemistry, we found that the expression levels of TbetaRII mRNA and protein in the epidermis of the forearm (sun-exposed) of the elderly were significantly lower than that of the upper-inner arm (sun-protected) skin of the same individual. In the epidermis, the expressions of Smad7 mRNA in both the intrinsically aged and photoaged skin of the elderly were higher than in the sun-protected skin of the young, and this was elevated in the photoaged epidermis. Decreased pSmad2 immunoreactivity was observed in the epidermis of photoaged forearm skin versus matched intrinsically aged skin. This decrease was also found in the epidermis of upper-inner arm skin of the elderly versus the young. These results suggest that the UV-induced down-regulation of TbetaRII and the concerted over-expression of Smad7 may trigger the inhibition of the TGF-beta-induced phosphorylation of Smad2.     

A novel promising therapy for skin aging: dermal multipotent stem cells against photoaged skin by activation of TGF-β/Smad and p38 MAPK signaling pathway

Skin photoaging, the most common skin damage, is caused by chronic UV irradiation. It is involved in the reduction, aging and apoptosis of fibroblasts (FBs) as well as the blockage of transforming growth factor-beta (TGF-β)/Smad and p38 mitogen-actived protein kinase (MAPK) signaling pathways. Dermal multipotent stem cells (dMSCs) are a population of adult stem cells derived from dermis in recent years. It has been confirmed that dMSCs can activate or differentiate into FBs to participate in wound healing by producing and expressing TGF-β and other cytokines. Considering the mechanism of skin photoaging and the role of dMSCs, we hold a hypothesis that dMSCs may be applied in skin photoaging by activating TGF-β/Smad and p38 MAPK signaling pathways, and then stimulating FBs to secrete and synthesize collagen or elastin, heightening the extracellular matrix, finally eliminating wrinkles and strengthening skin elasticity. These would provide a novel approach for anti-skin photoaging.     

Inhibition of type I procollagen synthesis by damaged collagen in photoaged skin and by collagenase-degraded collagen in vitro

Type I and type III procollagen are reduced in photodamaged human skin. This reduction could result from increased degradation by metalloproteinases and/or from reduced procollagen synthesis. In the present study, we investigated type I procollagen production in photodamaged and sun-protected human skin. Skin samples from severely sun-damaged forearm skin and matched sun-protected hip skin from the same individuals were assessed for type I procollagen gene expression by in situ hybridization and for type I procollagen protein by immunostaining. Both mRNA and protein were reduced ( approximately 65 and 57%, respectively) in photodamaged forearm skin compared to sun-protected hip skin. We next investigated whether reduced type I procollagen production was because of inherently reduced capacity of skin fibroblasts in severely photodamaged forearm skin to synthesize procollagen, or whether contextual influences within photodamaged skin act to down-regulate type I procollagen synthesis. For these studies, fibroblasts from photodamaged skin and matched sun-protected skin were established in culture. Equivalent numbers of fibroblasts were isolated from the two skin sites. Fibroblasts from the two sites had similar growth capacities and produced virtually identical amounts of type I procollagen protein. These findings indicate that the lack of type I procollagen synthesis in sun-damaged skin is not because of irreversible damage to fibroblast collagen-synthetic capacity. It follows, therefore, that factors within the severely photodamaged skin may act in some manner to inhibit procollagen production by cells that are inherently capable of doing so. Interactions between fibroblasts and the collagenous extracellular matrix regulate type I procollagen synthesis. In sun-protected skin, collagen fibrils exist as a highly organized matrix. Fibroblasts are found within the matrix, in close apposition with collagen fibers. In photodamaged skin, collagen fibrils are shortened, thinned, and disorganized. The level of partially degraded collagen is approximately 3.6-fold greater in photodamaged skin than in sun-protected skin, and some fibroblasts are surrounded by debris. To model this situation, skin fibroblasts were cultured in vitro on intact collagen or on collagen that had been partially degraded by exposure to collagenolytic enzymes. Collagen that had been partially degraded by exposure to collagenolytic enzymes from either bacteria or human skin underwent contraction in the presence of dermal fibroblasts, whereas intact collagen did not. Fibroblasts cultured on collagen that had been exposed to either source of collagenolytic enzyme demonstrated reduced proliferative capacity (22 and 17% reduction on collagen degraded by bacterial collagenase or human skin collagenase, respectively) and synthesized less type I procollagen (36 and 88% reduction, respectively, on a per cell basis). Taken together, these findings indicate that 1) fibroblasts from photoaged and sun-protected skin are similar in their capacities for growth and type I procollagen production; and 2) the accumulation of partially degraded collagen observed in photodamaged skin may inhibit, by an as yet unidentified mechanism, type I procollagen synthesis.     

Chronic ultraviolet B irradiation causes loss of hyaluronic acid from mouse dermis because of down-regulation of hyaluronic acid synthases

Remodeling of the dermal extracellular matrix occurs during photoaging. Here, the effect of repetitive UVB irradiation on dermal hyaluronic acid (HA) was examined. C57/BL6 mice were chronically (182 days) irradiated with UVB, and consecutive skin biopsies were collected during the irradiation period and afterward (300 and 400 days of age). UVB caused marked loss of HA from the papillary dermis and down-regulation of HA synthase 1 (HAS1), HAS2, and HAS3 mRNA expression. In contrast, hyaluronidases (HYAL) 1, HYAL2, and HA receptor CD44 were unchanged. Furthermore, transforming growth factor beta-1 (TGF-beta1) and TGF-beta1-receptor II expression were decreased in UVB-irradiated biopsies, and TGF-beta1 strongly induced HAS1 and HAS2 expression in cultured dermal fibroblasts. Therefore, TGF-beta1 might be one factor involved in UVB-induced down-regulation of HAS enzymes. In addition, total cell number and the percentage of proliferating fibroblasts in the papillary dermis of UVB-irradiated mice were decreased. Down-regulation of HAS2 by lentiviral overexpression of short hairpin RNA in vitro caused inhibition of HA synthesis, DNA synthesis, and migration of dermal fibroblasts. In conclusion, chronic UVB irradiation induces loss of HA from the dermis, thereby contributing to the quiescent phenotype of dermal fibroblasts.     

Regulation of type I procollagen and MMP-1 expression after single or repeated exposure to infrared radiation in human skin

Human skin is daily exposed to infrared (IR) radiation from natural sunlight. However, the effects of IR irradiation on collagen metabolism have not been investigated in human skin in vivo. Here, we examined whether single or repeated (three times a week for 4 weeks) exposure to IR irradiation changes the expressions of type I procollagen and interstitial collagenase (MMP-1). By using immunostaining, Western blotting, and semi-quantitative RT-PCR, we analyzed the protein and mRNA levels of type I procollagen and MMP-1 in young buttock skin. A single dose of IR to human skin increased the expression of type I procollagen within 24h, but did not change the expression of MMP-1. On the other hand, multiple IR doses reduced the expression of type I procollagen and increased the expression of MMP-1. We also found that TGF-betas may mediate type I procollagen synthesis in IR-irradiated human skin. Our results demonstrate that the regulations of the expressions of type I procollagen and MMP-1 differ in acute and chronically IR-irradiated skin. In particular, decreased collagen levels and increased MMP-1 levels in chronic IR-irradiated skin may be associated with connective tissue damage. Thus, we suggest that repeated exposure to IR irradiation might induce premature skin aging (photoaging) in human skin in vivo.     

Mice lacking Smad3 are protected against cutaneous injury induced by ionizing radiation

Transforming growth factor-beta (TGF-beta) plays a central role in the pathogenesis of inflammatory and fibrotic diseases, including radiation-induced fibrosis. We previously reported that mice null for Smad3, a key downstream mediator of TGF-beta, show accelerated healing of cutaneous incisional wounds with reduced inflammation and accumulation of matrix. To determine if loss of Smad3 decreases radiation-induced injury, skin of Smad3+/+ [wild-type (WT)] and -/- [knockout (KO)] mice was exposed to a single dose of 30 to 50 Gy of gamma-irradiation. Six weeks later, skin from KO mice showed significantly less epidermal acanthosis and dermal influx of mast cells, macrophages, and neutrophils than skin from WT littermates. Skin from irradiated KO mice exhibited less immunoreactive TGF-beta and fewer myofibroblasts, suggesting that these mice will have a significantly reduced fibrotic response. Although irradiation induced no change in the immunohistochemical expression of the TGF-beta type I receptor, the epidermal expression of the type II receptor was lost after irradiation whereas its dermal expression remained high. Primary keratinocytes and dermal fibroblasts prepared from WT and KO mice showed similar survival when irradiated, as did mice exposed to whole-body irradiation. These results suggest that inhibition of Smad3 might decrease tissue damage and reduce fibrosis after exposure to ionizing irradiation.     

Augmentation of UV-induced skin wrinkling by infrared irradiation in hairless mice

Skin aging can be divided into intrinsic aging and photoaging. Sunlight is a major cause of photoaging, and is composed of ultraviolet (UV) and infrared (IR) radiation. Although the effects of UV radiation on skin aging have been widely studied, little is known about the biological effects of IR on the photoaging process in human skin. We found that chronic IR treatment induced wrinkles in hairless mice, and augments UV-induced wrinkle formation and UV-induced skin thickening in hairless mice. Histologically, we found that IR treatment augments UV-induced epidermal and dermal thickening, and that UV-induced increases of collagen and elastic fibers in dermis. Moreover, chronic IR treatment increased MMP-3 and MMP-13 mRNA expressions significantly in hairless mouse skin and augmented UV-induced MMP-3 and MMP-13 mRNA expressions and UV-induced MMP-2 and MMP-9 activities. From these results, we demonstrate that IR alone induces skin wrinkling and augments UV-induced wrinkle formation. Taken together, we suggest that IR plays an important role in the development of photoaging.     

Enhanced expression of cylooxygenase-2 by UV in aged human skin in vivo

Prostaglandins (PGs) induced by UV may play important roles in UV-induced inflammation, photocarcinogenesis, and photoaging processes in human skin. The age-related PGE2 production and cyclooxygenase-2 (COX-2) expression in the human skin in vivo remain unclear. The purpose of this study was to examine the influence of aging on UV-induced PGE2 production and COX-2 expression in human skin in vivo. We found that aged human skin produces higher amounts of PGE2 than young skin, when exposed to UV. The inductions of COX-2 mRNA and protein by UV in aged skin were higher than those in the young skin, whereas COX-1 mRNA expression remained unchanged. Aged human macrophage expressed higher amounts of PGE2 and COX-2 protein constitutively, and also induced these species after LPS treatment more so than young cells. Our data suggest that skin aging may increase susceptibility to the development of skin cancer and photoaging, by enhanced PGE2 and COX-2 expression due to UV in human skin in vivo.     

Expression of TGF-beta1, TbetaRII and Smad4 in colorectal carcinoma

BACKGROUND: Many colorectal carcinomas are resistant to the growth inhibitory response of transforming growth factor-beta (TGF-beta) due to alterations of components along the TGF-beta signaling pathway. The aim of this study was to examine the expression of TGF-beta1, TbetaRII and Smad4 in human colorectal carcinoma and their relationships with cancer growth. METHODS: Immunohistochemistry and in situ hybridization were performed in 38 cases of colorectal carcinoma. RESULTS: Intense signal for TGF-beta1 protein and TGF-beta1 mRNA were found in 71.1% (27/38) and 77.8% (21/27) of colorectal carcinoma, respectively. Intensive TbetaRII mRNA were detected only in 40% (11/27) cancer tissues (p<0.05). 65.8% (25/38) of colorectal carcinoma displayed decreased expression in TbetaRII immunoreactivity staining (p<0.05). Smad4 protein and Smad4 mRNA were reduced in 63.2% (24/38) and 63% (17/27) of tumors, respectively. Smad4 expression was related to tumor differentiation and Duke's stage (p<0.05). Furthermore, TGF-beta1-positive tumors with lymph node metastasis preferentially had significant reduced Smad4 expression (p<0.05). CONCLUSIONS: Down-regulation of TbetaRII as well as the over-expression of TGF-beta1 play a possible role for the escape of colorectal carcinoma from TGF-beta-mediated growth inhibition. Reduced Smad4 is associated with malignancy and progression of colorectal carcinoma.     

Interference with transforming growth factor-beta/ Smad3 signaling results in accelerated healing of wounds in previously irradiated skin

Transforming growth factor (TGF)-beta regulates many aspects of wound repair including inflammation, chemotaxis, and deposition of extracellular matrix. We previously showed that epithelialization of incisional wounds is accelerated in mice null for Smad3, a key cytoplasmic mediator of TGF-beta signaling. Here, we investigated the effects of loss of Smad3 on healing of wounds in skin previously exposed to ionizing radiation, in which scarring fibrosis complicates healing. Cutaneous wounds made in Smad3-null mice 6 weeks after irradiation showed decreased wound widths, enhanced epithelialization, and reduced numbers of neutrophils and myofibroblasts compared to wounds in irradiated wild-type littermates. Differences in breaking strength of wild-type and Smad3-null wounds were not significant. As shown previously for neutrophils, chemotaxis of primary dermal fibroblasts to TGF-beta required Smad3, but differentiation of fibroblasts to myofibroblasts by TGF-beta was independent of Smad3. Previous irradiation-enhanced induction of connective tissue growth factor mRNA in wild-type, but not Smad3-null fibroblasts, suggested that this may contribute to the heightened scarring in irradiated wild-type skin as demonstrated by Picrosirius red staining. Overall, the data suggest that attenuation of Smad3 signaling might improve the healing of wounds in previously irradiated skin commensurate with an inhibition of fibrosis.     

IL-1 receptor antagonist attenuates MAP kinase/AP-1 activation and MMP1 expression in UVA-irradiated human fibroblasts induced by culture medium from UVB-irradiated human skin keratinocytes

Solar UV light comprises UVB wavelengths (290-320 nm) and UVA wavelengths (320-400 nm). UVB radiation reaches the epidermis and, to a lesser extent, the upper part of the dermis, while UVA radiation penetrates more deeply into human skin. Existing studies have demonstrated that UV-irradiated epidermal keratinocytes release cytokines that indirectly promote MMP-1 production in dermal fibroblasts. In this study, we first investigated the effect of IL-1 on MAPK activity, c-Jun and c-Fos mRNA expression, and MMP-1 and MMP-2 production in UVA-irradiated human dermal fibroblasts. The results showed that UVA irradiation dose-dependently increased MMP-1 but not MMP-2 production in human skin fibroblasts. IL-1alpha and IL-1beta promoted MMP-1 but not MMP-2 production in UVA-irradiated fibroblasts. Both IL-1alpha and IL-1beta activated MAP kinase, significantly elevating c-Jun and c-Fos mRNA expression. We then investigated the indirect effect of UVB-irradiated keratinocyte culture medium on MMP-1 production in UVA-irradiated primary cultured human dermal fibroblasts and the effect of IL-1Ra. The results showed that cell culture medium from UVB-irradiated keratinocytes increased MMP-1 production in UVA-irradiated fibroblasts, and IL-1Ra dose-dependently inhibited MMP-1 production. IL-1Ra dose-dependently inhibited c-Jun mRNA expression of fibroblasts with no significant effect on c-Fos mRNA expression. These results demonstrate that UVB-irradiated keratinocytes promoted MMP-1 production in UVA-irradiated fibroblasts in a paracrine manner while IL-1Ra reduced MMP-1 production through inhibiting c-Jun mRNA expression. Collectively, our data suggest that IL-1 plays an important role in the dermal collagen degradation associated with UV-induced premature aging of the skin and IL-1Ra may be applied for the prevention and treatment of photoaging.     

Kanglaite attenuates UVB-induced down-regulation of aquaporin-3 in cultured human skin keratinocytes

Ultraviolet (UV) radiation plays an important role in the pathogenesis of skin photoaging. Depending on the wavelength of UV, the epidermis is affected primarily by UVB. One major characteristic of photoaging is the dehydration of the skin. Membrane-inserted water channels (aquaporins) are involved in this process. In this study we demonstrated that UVB radiation induced aquaporin-3 (AQP3) down-regulation in cultured human skin keratinocytes. Kanglaite is a mixture consisting of extractions of Coix Seed, which is an effective anti-neoplastic agent and can inhibit the activities of protein kinase?C and NF-κB. We demonstrated that Kanglaite inhibited UVB-induced AQP3 down-regulation of cultured human skin keratinocytes. Our findings provide a potential new agent for anti-photoaging. The related molecular mechanisms remain to be further elucidated.     

Expression of transforming growth factor-beta type I and type II receptors is altered in rat lungs undergoing bleomycin-induced pulmonary fibrosis

Transforming growth factor-beta (TGF-beta) is a family of autocrine/paracrine/endocrine cytokines involved in controlling cell growth and extracellular matrix metabolism. TGF-beta exerts its biological effects via binding to type I (TbetaRI) and type II (TbetaRII) receptors. To gain insight into the possible role of TGF-beta receptors in the pathogenesis of pulmonary fibrosis, we investigated the expression of TGF-beta receptors and their ligands in a bleomycin-induced model of pulmonary fibrosis. We found that the expression of both TbetaRI and TbetaRII was altered in rat lungs during pulmonary fibrosis induced by bleomycin. The increase in TbetaRI mRNA level was evident after 3 days of bleomycin administration, and TbetaRI mRNA continually increased for over 12 days after bleomycin instillation, whereas TbetaRII mRNA declined at day 3 post bleomycin instillation and then increased during the reparative phase of lung injury (days 8 and 12). The immunoreactivity for both TbetaRI and TbetaRII was detected in the cells of the interstitium, the epithelium, and the blood vessels of normal rat lungs. In bleomycin-induced pulmonary fibrosis, an extensive immunostaining for TbetaRI and TbetaRII was present in the cells at the sites of injury and active fibrosis. These results demonstrate that the expression of TGF-beta type I and type II receptors was altered during pulmonary fibrosis, suggesting that the TGF-beta signal transduction pathway may be involved in the pathogenesis of lung fibrosis.     

Inhibition of p38 mitogen-activated protein kinase and transforming growth factor-beta1/Smad signaling pathways modulates the development of fibrosis in adriamycin-induced nephropathy

Inflammation and fibrogenesis are the two determinants of the progression of renal fibrosis, the common pathway leading to end-stage renal disease. The p38 mitogen-activated protein kinase (MAPK) and transforming growth factor (TGF)-beta1/Smad signaling pathways play critical roles in inflammation and fibrogenesis, respectively. The present study examined the beneficial renoprotective effect of combination therapy using the p38 MAPK pathway inhibitor (SB203580) and a TGF-beta receptor I (ALK5) inhibitor (ALK5I) in a mouse model of adriamycin (ADR) nephrosis. The p38 MAPK and TGF-beta1/Smad2 signaling pathways were activated in ADR-induced nephropathy in a sequential time course manner. Two weeks after ADR injection, the combined administration of SB203580 (1 mg/kg/24 hours) and ALK5I (1 mg/kg/24 hours) markedly reduced p38 MAPK and Smad2 activities. Moreover, the co-administration of SB203580 and ALK5I to ADR-injected mice resulted in a down-regulation of total and active TGF-beta1 production, reduced myofibroblast accumulation, and decreased expression of collagen type IV and fibronectin. In these mice, retardation in the development of glomerulosclerosis and interstitial fibrosis was observed. In conclusion, although p38 MAPK and TGF-beta1/Smad signaling pathways are distinct they coordinate the progression of renal fibrosis in ADR nephrosis. The co-administration of a p38 MAPK inhibitor and an ALK5 inhibitor may have potential applications in the treatment of renal fibrosis.     

UV radiation and prostaglandin E2 up-regulate vascular endothelial growth factor (VEGF) in cultured human fibroblasts

OBJECTIVE AND DESIGN: Exposure to UV radiation is responsible for skin erythema and inflammation. PGE2 is an important inflammatory mediator involved in this process and vascular endothelial growth factor (VEGF) is a potent vascular permeability factor mainly produced by epidermal keratinocytes. This study was aimed at determining whether UVB/A1 radiation and prostaglandin E2 (PGE2) could modulate the production of VEGF by cultured dermal human fibroblasts (HF) in comparison to keratinocytes (HK). MATERIALS AND METHODS: The skin cells derived from foreskin, were cultured in defined medium before treatment by either UVB/A1 radiation, or stimulation by addition of PGE2 (10(-8) to 10(-5) M). The expression of VEGF in cultured fibroblasts and keratinocytes was evaluated at the mRNA (RT-PCR) and protein levels (ELISA). RESULTS: The basal level of VEGF was lower in HF than in HK. Both UVB and UVA1 radiation strongly up-regulated VEGF mRNA and protein in HF whereas UVB but not UVA1 radiation induced a VEGF increase in HK only at the protein level. UVA1, when associated with UVB radiation, showed an additive effect on VEGF secretion in HF but not in HK. PGE2 increased in a dose-dependent manner the expression of VEGF in HF but not in HK. Indomethacin as well as the antioxidant alpha-tocopherol did not reduce UV-induced enhanced secretion of VEGF by both fibroblasts and keratinocytes whereas pyrolidine dithiocarbamate exerted an inhibition of this overexpression. CONCLUSIONS: These results indicate different signaling pathways in the PGE2 and UV-induced regulation of VEGF in dermal fibroblasts and epidermal keratinocytes. They also suggest a role for VEGF from both fibroblasts and keratinocytes in the UV-induced erythema, independent of PGE2. A dermal overexpression of VEGF by fibroblasts from UV-irradiated skin may contribute to dilated microvasculature, a feature of skin photoaging and more generally, to a more permissive stroma to tumor formation than unexposed skin.