Peroxidized cholesterol-induced matrix metalloproteinase-9 activation and its suppression by dietary beta-carotene in photoaging of hairless mouse skin

The activation of matrix metalloproteinase (MMP)-9 leading to the formation of wrinkle and sagging of skin is an essential step in the skin photoaging on exposure to ultraviolet A (UVA). This study attempted to elucidate the role of peroxidized cholesterol including cholesterol hydroperoxides (Chol-OOHs), primary products of lipid peroxidation in biomembranes, in MMP-9 activation and the effect of dietary beta-carotene in MMP-9 activation. Hairless mice were subjected to periodic UVA irradiation for 8 weeks. The amount of peroxidized cholesterol detected as total hydroxycholesterol in the skin was increased significantly by the exposure. The activity and protein level of MMP-9 were elevated with wrinkling and sagging formation. MMP-9 activity was also enhanced by the intracutaneous injection of Chol-OOHs into the mouse skin. Adding beta-carotene to the diet of the mice during the period of irradiation suppressed the activity and expression of MMP-9 as well as the wrinkling and sagging formation. The amount of cholesterol 5alpha-hydroperoxide, a singlet molecular oxygen oxygenation-specific peroxidized cholesterol, was significantly lowered by the addition of beta-carotene to the diet. These results strongly suggest that Chol-OOHs formed on exposure to UVA contribute to the expression of MMP-9, resulting in photoaging. Dietary beta-carotene prevents the expression of MMP-9, at least partly, by inhibiting photodynamic action involved in the formation of Chol-OOHs.     

Protective effects of silkworm hemolymph extract and its fractions on UV-induced photoaging

Ultraviolet (UV) irradiation induces skin photoaging by generating reactive oxygen species (ROS). ROS caused by UV-irradiation results in loss of skin cells and degradation of extracellular matrix. A number of antioxidants have been chemically synthesized or naturally extracted to prevent ROS-mediated skin photoaging. In our previous work, silkworm hemolymph extract (SHEX) was prepared, and its antioxidant activity was tested by free radical-scavenging assay. This study assessed the protective effects of SHEX on UV-induced photoaging of human immortalized keratinocytes (HaCaT). UVA (365 nm)-induced ROS generation was inhibited by supplementation of silkworm hemolymph (SH). Treatment with SHEX prepared by boiling SH inhibited death of HaCaT cells caused by UVB (315 nm) and UVA irradiation in a dose-dependent manner. Seven fractions were obtained by separating SHEX by gel permeation chromatography and the antioxidant activity of the fractions was examined. The fraction showing the highest protective efficacy on UV-induced cell damage corresponded to the lutein-containing fraction isolated in our previous study. Moreover, the SHEX fraction suppressed the expression of MMP-1 (matrix metalloproteinase-1), a matrix-degrading enzyme, suggesting that the active constituent of SHEX has the potential to inhibit skin photoaging. These results suggest that SHEX can be developed as a dietary and cosmetic supplement for prevention of skin photoaging.     

UVA-mediated downregulation of MMP-2 and MMP-9 in human epidermal keratinocytes

While human dermal fibroblasts increase the expression and secretion of distinct matrix metalloproteinases (MMPs) in response to ultraviolet (UV) irradiation, much less is known about regulation of MMPs with regard to normal human epidermal keratinocytes (NHEK). In this in vitro study, the effect of ultraviolet A (UVA) irradiation on gelatinase expression and secretion by NHEK was investigated. Irradiation of NHEK with non-toxic doses of UVA resulted in a dose-dependent downregulation of MMP-2 (gelatinase A) and MMP-9 (gelatinase B). A single dose of 30JUVA/cm(2) lowered MMP-2 activity to 26% and MMP-9 activity to 33% compared with mock-irradiated cells at 24h after irradiation. Downregulation of MMP-2 and MMP-9 steady-state mRNA levels was observed at 4h after UVA irradiation. The inhibitory effect of UVA on gelatinases was mediated by UVA-generated singlet oxygen (1O(2)). These findings suggest an inverse response to UVA irradiation in NHEK than in fibroblasts.     

Toll-like receptor adaptor signaling molecule MyD88 on intervertebral disk homeostasis: in vitro, ex vivo studies

MyD88 is an adapter protein that links toll-like receptors (TLRs) and Interleukin-1 receptors (IL-1Rs) with downstream signaling molecules. The MyD88 has been found to be an essential mediator in the development of osteoarthritis in articular cartilage. However, the role of the MyD88 pathway has yet to be elucidated in the intervertebral disk (IVD). Using in vitro techniques, we analyzed the effect of MyD88 pathway-specific inhibition on the potent inflammatory and catabolic mediator LPS and IL-1 in bovine and human nucleus pulposus (NP) cells by assessing matrix-degrading enzyme expression, including matrix metalloproteases (MMPs) and a disintegrin-like and metalloprotease with thrombospondin motifs (ADAMTS family). We also analyzed inhibition of MyD88 in the regulation of inducible nitric oxide synthase and TLR-2. Finally, we used an ex vivo organ culture model to assess the effects of MyD88 inhibitor (MyD88i) on catabolic factor-induced disk degeneration in mice lumbar disks. In bovine NP cells, MyD88i potently antagonizes LPS- or IL-1-mediated induction of cartilage-degrading enzyme production, including MMP-1, MMP-13, ADAMTS-4, and ADAMTS-5. MyD88i also attenuates the LPS- or IL-1-mediated induction of iNOS and TLR-2 gene expression. Our ex vivo findings reveal inhibition of MyD88 via counteraction of IL-1-mediated proteoglycan depletion. The findings from this study demonstrate the potent anti-inflammatory and anti-catabolic effects of inhibition of MyD88 pathway inhibition on IVD homeostasis, suggesting a potential therapeutic benefit of a MyD88i in degenerative disk disease in the future.     

Sunscreens with an absorption maximum of > or =360 nm provide optimal protection against UVA1-induced expression of matrix metalloproteinase-1, interleukin-1, and interleukin-6 in human dermal fibroblasts.

UVA1-induced expression of matrix metalloproteinase-1 (MMP-1) is mediated by an autocrine mechanism involving the cytokines interleukin-1 and -6 (IL-1 and IL-6). The subsequent degradation of collagen fibers is thought to be the main cause of skin wrinkling. As it is currently not known which wavelengths within the UVA1 range are responsible for these effects, we have assessed 5 UVA1 filters (experimental filters HRH21328 and HRH22127, butyl methoxydibenzoylmethane (BMDM), diethylaminohydroxybenzylbenzoic acid hexyl ester (DHBB) and anisotriazine) with different absorption maxima for their capacity to protect against UVA1-induced MMP-1 expression. To test the efficacy of these hydrophobic filters in a cell culture system, UVA1 irradiation of primary human fibroblasts was performed through a quartz microplate filled with ethanolic solutions of the UVA filters placed on top of the cell microplate. Inhibition of UVA1-induced gene expression was detected by real time RT-PCR. The efficacy to protect against UVA1-induced MMP-1 expression was wavelength dependent: the protection by HRH22127 was best, followed by HRH21328, DHBB, BMDM, and anisotriazine. In addition, HRH22127 and HRH 21328 both significantly inhibited UVA1-induced expression of IL-1alpha and IL-6 with HRH21238 being superior to HRH22127. These studies indicate that UVA1 filters with a maximum absorption at > or =360 nm are most effective in preventing UVA1 radiation-induced MMP-1, IL-1alpha, and IL-6 expression pointing towards a critical role for effective filtering beyond > or =360 nm for protection against UVA1-induced photoaging.     

UVA-mediated down-regulation of MMP-2 and MT1-MMP coincides with impaired angiogenic phenotype of human dermal endothelial cells

UVA irradiation, dose-dependently (5-20 J/cm2), was shown to impair the morphogenic differentiation of human microvascular endothelial cells (HMECs) on Matrigel. Parallely, UVA down-regulated the expression of MMP-2 and MT1-MMP, both at the protein and the mRNA levels. On the contrary, the production of MMP-1 and TIMP-1 by HMECs increased following UVA treatment. The inhibitory effect of UVA on MMP expression and pseudotubes formation was mediated by UVA-generated singlet oxygen (1O2). The contribution of MT1-MMP, but not TIMP-1, to the regulation of HMECs' angiogenic phenotype following UVA irradiation was suggested using elastin-derived peptides and TIMP-1 blocking antibody, respectively.     

Overexpression of phospholipid-hydroperoxide glutathione peroxidase in human dermal fibroblasts abrogates UVA irradiation-induced expression of interstitial collagenase/matrix metalloproteinase-1 by suppression of phosphatidylcholine hydroperoxide-mediated NFkappaB activation and interleukin-6 release

Phospholipid-hydroperoxide glutathione peroxidase (PHGPx) exhibits high specific activity in reducing phosphatidylcholine hydroperoxides (PCOOHs) and thus may play a central role in protecting the skin against UV irradiation-triggered detrimental long term effects like cancer formation and premature skin aging. Here we addressed the role of PHGPx in the protection against UV irradiation-induced expression of matrix metalloproteinase-1 (MMP-1). For this purpose, we created human dermal fibroblast cell lines overexpressing human PHGPx. Overexpression led to a significant increase in PHGPx activity. In contrast to a maximal 4.5-fold induction of specific MMP-1 mRNA levels in vector-transfected cells at 24 h after UVA irradiation, no MMP-1 induction occurred at any studied time point after UVA treatment of PHGPx-overexpressing fibroblasts. As interleukin-6 (IL-6) was earlier shown to mediate the UVA induction of MMP-1, we studied whether PHGPx overexpression might interfere with the NFkappaB-mediated IL-6 induction and downstream signaling. Using transient transfections of IL-6 promoter constructs containing NFkappaB binding sites, we observed a high induction of the reporter gene luciferase in vector-transfected control cells and a significantly lower induction in PHGPx-overexpressing fibroblasts following UVA irradiation. Consistently both UVA irradiation and treatment of fibroblasts with PCOOHs led to phosphorylation and nuclear translocation of the p65 subunit, whereas cells overexpressing PHGPx exhibited impaired NFkappaB activation, p65 phosphorylation, and nuclear translocation. In line with this, the PHGPx-overexpressing fibroblasts showed a reduced constitutive and UVA irradiation-induced IL-6 release. After incubating PHGPx-overexpressing cells with PCOOHs a reduced induction of IL-6 was observed. This together with the suppression of UVA irradiation-induced IL-6 release in the presence of Trolox, a chain breaker of PCOOH-initiated lipid peroxidation, indicates that UVA irradiation-induced PCOOHs and subsequent lipid peroxides initiate the NFkappaB-mediated induction of IL-6, which mediates the induction of MMP-1. Our finding is particularly relevant in light of the already available small molecule mimetics of PHGPx.     

MC1R expression in HaCaT keratinocytes inhibits UVA-induced ROS production via NADPH oxidase- and cAMP-dependent mechanisms

Ultraviolet A (UVA) radiations are responsible for deleterious effects, mainly due to reactive oxygen species (ROS) production. Alpha-melanocyte stimulating hormone (α-MSH) binds to melanocortin-1 receptor (MC1R) in melanocytes to stimulate pigmentation and modulate cutaneous inflammatory responses. MC1R may be induced in keratinocytes after UV exposure. To investigate the effect of MC1R signaling on UVA-induced ROS (UVA-ROS) production, we generated HaCaT cells that stably express human MC1R (HaCaT-MC1R) or the Arg151Cys (R(151)C) non-functional variant (HaCaT-R(151)C). We then assessed ROS production immediately after UVA exposure and found that: (1) UVA-ROS production was strongly reduced in HaCaT-MC1R but not in HaCaT-R(151)C cells compared to parental HaCaT cells; (2) this inhibitory effect was further amplified by incubation of HaCaT-MC1R cells with α-MSH before UVA exposure; (3) protein kinase A (PKA)-dependent NoxA1 phosphorylation was increased in HaCaT-MC1R compared to HaCaT and HaCaT-R(151)C cells. Inhibition of PKA in HaCaT-MC1R cells resulted in a marked increase of ROS production after UVA irradiation; (4) the ability of HaCaT-MC1R cells to produce UVA-ROS was restored by inhibiting epidermal growth factor receptor (EGFR) or extracellular signal-regulated kinases (ERK) activity before UVA exposure. Our findings suggest that constitutive activity of MC1R in keratinocytes may reduce UVA-induced oxidative stress via EGFR and cAMP-dependent mechanisms.     

Photoprotective potential of lycopene,beta-carotene,vitamin E,vitamin C and carnosic acid in UVA-irradiated human skin fibroblasts

The photoprotective potential of the dietary antioxidants vitamin C, vitamin E, lycopene, beta-carotene, and the rosemary polyphenol, carnosic acid, was tested in human dermal fibroblasts exposed to ultraviolet-A (UVA) light. The carotenoids were prepared in special nanoparticle formulations together with vitamin C and/or vitamin E. Nanoparticle formulations, in contrast to dimethylsulphoxide, stablized lycopene in the cell culture medium and allowed efficient cellular uptake. The presence of vitamin E in the formulation further increased the stability and cellular uptake of lycopene. UVA irradiation of the human skin fibroblasts led to a 10-15-fold rise in metalloproteinase 1 (MMP-1) mRNA. This rise was suppressed in the presence of low microM concentrations of vitamin E, vitamin C, or carnosic acid but not with beta-carotene or lycopene. Indeed, in the presence of 0.5-1.0 microM beta-carotene or lycopene, the UVA-induced MMP-1 mRNA was further increased by 1.5-2-fold. This increase was totally suppressed when vitamin E was included in the nanoparticle formulation. Heme-oxygenase 1 (HO-1) mRNA expression was strongly induced by UVA irradiation but none of the antioxidants inhibited this effect at the concentrations used in this study. Indeed, beta-carotene or lycopene (0.5-1.0 microM) led to a further 1.5-fold rise in the UVA-induced HO-1 mRNA levels. In conclusion, vitamin C, vitamin E, and carnosic acid showed photoprotective potential. Lycopene and beta-carotene did not protect on their own but in the presence of vitamin E, their stability in culture was improved and the rise in MMP-1 mRNA expression was suppressed, suggesting a requirement for antioxidant protection of the carotenoids against formation of oxidative derivatives that can influence the cellular and molecular responses.     

Monocyte membrane type 1-matrix metalloproteinase. Prostaglandin-dependent regulation and role in metalloproteinase-2 activation

Membrane type 1-matrix metalloproteinase (MT1-MMP)-mediated activation of MMP-2 is thought to be important in the proteolysis of extracellular matrix in pathological events in which monocytes/macrophages are found. Here we report on the induction and regulation of human monocyte MT1-MMP and its role in MMP-2 activation. Activation of monocytes by lipopolysaccharide resulted in the induction of MT1-MMP mRNA and protein that was suppressed by inhibitors of prostaglandin synthesis (indomethacin), adenylyl cyclase (SQ 22536), and protein kinase A (Rp-cAMPs). Suppression of MT1-MMP by indomethacin and SQ 22536 was reversed by prostaglandin E(2) and dibutyryl cyclic AMP, respectively, demonstrating that induction of monocyte MT1-MMP is regulated through a prostaglandin-cAMP pathway. Functional analysis revealed that pro-MMP-2 in the supernatants from human bone marrow stromal fibroblasts, normal male-derived fibroblasts and melanoma cells (A2058) was converted to active MMP-2 when cultured with activated but not control monocytes. Antibodies against MT1-MMP blocked the activation of MMP-2. Tissue inhibitor of metalloproteinase-2 regulation of MMP-2 activation was shown through the addition of varying amounts of recombinant tissue inhibitor of metalloproteinase-2 with pro-MMP-2 to MT1-MMP-expressing monocytes. These findings demonstrate that activated monocytes express functionally active MT1-MMP that may play a significant role in the activation of MMP-2 produced by other cells and as such influence developmental and pathological conditions.     

The effect of beta-carotene on the expression of interleukin-6 and heme oxygenase-1 in UV-irradiated human skin fibroblasts in vitro.

beta-Carotene is discussed as an anti-oxidant micronutrient and singlet oxygen quencher in human skin, protecting against UV light-induced damage. However, we recently demonstrated that beta-carotene has a pro-oxidant potential in cultured human skin fibroblasts because it enhances the UVA induction of heme oxygenase-1 (HO-1). Herein, we further show that beta-carotene also strongly promotes the UVA induction of pro-inflammatory interleukin-6 (IL-6) in skin fibroblasts in vitro. Singlet oxygen quencher sodium azide abrogated up-regulation of IL-6, and likewise also of HO-1. In UVB-irradiated cells, beta-carotene did not modulate levels of IL-6 and HO-1. The observed effects might be relevant for UV-induced inflammatory processes.     

Effect of a novel ascorbic derivative, disodium isostearyl 2-O-L-ascorbyl phosphate, on normal human dermal fibroblasts against reactive oxygen species

The novel amphiphilic vitamin C derivative disodium isostearyl 2-O-L-ascorbyl phosphate (VCP-IS-2Na), which has a C(18) alkyl chain attached to the stable ascorbate derivative sodium L-ascorbic acid 2-phosphate (VCP-Na), was evaluated for reduction of cell damage induced by oxidative stress, ultraviolet A (UVA), ultraviolet B (UVB), and H(2)O(2); stimulation of collagen synthesis against UVA irradiation; and inhibition of matrix metalloproteinase-1 (MMP-1) activity induced by UVA in human normal dermal fibroblasts. VCP-IS-2Na pretreatment resulted in significant protection against cell damage induced by UVB, UVA, and H(2)O(2). The amount of type I collagen following UVA irradiation was increased by treatment with VCP-IS-2Na in a concentration-dependent manner. These effects of VCP-IS-2Na were superior to those of L-ascorbic acid (vitamin C, VC) and VCP-Na. On the other hand, VCP-IS-2Na suppressed 65% of the excess MMP-1 irradiated UVA, and VC and VCP-Na slightly suppressed it.     

Regulation of gelatinase-A (MMP-2) production by ovine intervertebral disc nucleus pulposus cells grown in alginate bead culture by Transforming Growth Factor-beta(1)and insulin like growth factor-I.

The aim of this study was to gain information relevant to disc repair processes. Limited degradation of the collagen matrix by matrix metalloproteases (MMPs) may facilitate the loosening of cell-cell and cell-matrix interactions within the injured intervertebral disc (IVD) to favour the penetration of blood vessels and migration of fibroblasts into the defect to promote repair processes. Gelatinase A (MMP-2) has a particularly important role to play in angiogenesis, in the present study we investigated the in vitro regulation of MMP-2 by Transforming Growth Factor-beta 1 (TGF-beta 1) and Insulin-like Growth Factor-1 (beta IGF-I) in cells from the nucleus pulposus (NP) of the ovine IVD. Ovine NP cells were grown in alginate bead cultures in complete medium (10% foetal calf serum) for 7 days, established in serum-free conditions for 24 h, then stimulated with TGF-beta 1 (0.1 or 10 ng/ml) or IGF-I (2 or 50 ng/ml) +/-Concanavalin A (20 microg/ml) for an additional 48 h. Conditioned medium was examined for matrix metalloproteases using gelatin zymography, Tissue Inhibitor of Metalloproteinase 2 (TIMP-2) and Membrane Type 1 Matrix Metalloproteinase (MT1-MMP) were immunolocalised in beads. Pro (72 kDa) and active (59 kDa) MMP-2 were the major gelatinolytic MMPs detected in control cultures, the TGF-beta 1 and IGF-I treatments significantly decreased levels of the active MMP-2, inclusion of Concanavalin A resulted in a complete reversal of this trend with IGF-I, and to a lesser extent with TGF-beta 1. Cell surface levels of TIMP-2 and MT1-MMP were decreased by the TGF-beta 1 treatment while IGF-I only appeared to decrease TIMP-2 expression. The findings of this study provide some insight as to why dense avascular connective tissues such as the intervertebral disc have such a poor healing potential.