Curcumin inhibits UVB‐induced matrix metalloproteinase‐1/3 expression by suppressing the MAPK‐p38/JNK pathways in human dermal fibroblasts

Curcumin (diferuloylmethane) is a polyphenol derived from turmeric (Curcuma longa), which is commonly used as a spice. Recent studies have shown that curcumin has a wide range of pharmacological activities, including anticarcinogenic, antioxidant, anti‐inflammatory and antiangiogenic activities. However, the antiphotoageing effects of curcumin have yet to be characterized. In this study, we investigated the inhibitory effects of curcumin on matrix metalloproteinase (MMP)‐1 and MMP‐3 expression in human dermal fibroblast cells. Western blot analysis revealed that curcumin inhibited ultraviolet (UV) B‐induced MMP‐1 and MMP‐3 expression. Furthermore, curcumin significantly blocked UVB‐induced reactive oxygen species generation in fibroblasts. Curcumin treatment significantly blocked the UVB‐induced activation of nuclear factor (NF)‐κB and activator protein (AP)‐1. Additionally, curcumin strongly repressed the UVB‐induced phosphorylation of p38 and c‐Jun N‐terminal kinase. Curcumin prevented UVB‐induced MMP expression through mitogen‐activated protein kinase/NF‐κB inhibition and AP‐1 activation. In conclusion, curcumin may be useful for preventing and treating skin photoageing.     

NADPH oxidase is a novel target of delphinidin for the inhibition of UVB‐induced MMP‐1 expression in human dermal fibroblasts

We investigated the reported antiphotoaging effects of the major anthocyanidin delphidin and sought to identify its specific molecular target during UVB‐induced MMP‐1 expression. Delphinidin treatment significantly inhibited UVB‐induced MMP‐1 expression in primary cultured human dermal fibroblasts (HDF), an effect associated with the suppression of MKK4‐JNK1/2, MKK3/6‐p38 and MEK‐ERK1/2 phosphorylation. Further investigation revealed that delphinidin significantly inhibited UVB‐induced ROS production and NOX activity. Interestingly, the inhibitory effect of delphinidin on UVB‐induced NOX activity was stronger than that of apocynin, a pharmaceutical NOX inhibitor. Fractioned cell analysis results using a Western blot assay showed that this effect occurred through the inhibition of UVB‐induced P47^phox (a NOX subunit) translocation from the cytosol to the membrane. Pull down assays demonstrated that delphinidin binds directly to P47^phox in vitro. Collectively, our results suggest that delphinidin targets NOX, resulting in the suppression of UVB‐induced MMP‐1 expression in human dermal fibroblasts.     

MMP‐10 (Stromelysin‐2) and MMP‐21 in human and murine squamous cell cancer

Abstract: The squamous cell cancers (SCC) of renal transplant recipients are more aggressive and metastasize earlier than those of the non‐immunocompromised population. Matrix metalloproteinases (MMPs) have a central role in tumor initiation, invasion and metastasis. Our aim was to compare the expression of MMPs‐10, ‐12 and ‐21 in SCCs from immunosuppressed (IS) and control patients and the contribution of MMPs‐10 and ‐21 to SCC development in the FVB/N‐Tg(KRT5‐Nfkbia)3Rto mouse line. Immunohistochemical analysis of 25 matched pairs of SCCs, nine of Bowen’s disease and timed back skin biopsies of mice with selective inhibition of Rel/NF‐κB signalling were performed. Semiquantitatively assessed stromal MMP‐10 expression was higher (P = 0.009) in the control group when compared with IS patients. Tumor cell‐derived MMP‐10, ‐12 and ‐21 expression did not differ between the groups but stromal fibroblasts of the control SCCs tended to express MMP‐21 more abundantly. MMP‐10 expression was observed already in Bowen’s disease while MMP‐21 was absent. MMP‐10 and ‐21 were present in inflammatory or stromal cells in ageing mice while dysplastic keratinocytes and invasive cancer were negative. Our results suggest that MMP‐10 may be important in the initial stages of SCC progression and induced in the stroma relating to the general host‐response reaction to skin cancer. MMP‐21 does not associate with invasion of SCC but may be involved in keratinocyte differentiation.     

S‐allyl cysteine inhibits TNF‐α‐induced inflammation in HaCaT keratinocytes by inhibition of NF‐ κB‐dependent gene expression via sustained ERK activation

Tumor necrosis factor‐α (TNF‐α)‐induced keratinocyte inflammation plays a key role in the pathogenesis of multiple inflammatory skin diseases. Here we investigated the anti‐inflammatory effect of S‐allyl cysteine (SAC) on TNF‐α‐induced HaCaT keratinocyte cells and the mechanism behind its anti‐inflammatory potential. SAC was found to inhibit TNF‐α‐stimulated cytokine expression. Further, SAC was found to inhibit TNF‐α‐induced activation of p38, JNK and NF‐κB pathways. Interestingly, SAC was found to differentially regulate ERK MAP kinase in cells. TNF‐α‐induced transient ERK activation and SAC treatment resulted in sustained ERK activation both in the presence and absence of TNF‐α. Additionally, SAC failed to inhibit the TNF‐α‐induced expression of the pro‐inflammatory cytokines TNF‐α and IL‐1β when cells were treated with the MEK inhibitor PD98059, suggesting that the anti‐inflammatory effect of SAC is via sustained activation of the ERK pathway. Since ERK activation has been reported to negatively regulate NF‐κB‐driven gene expression and we find that SAC activates ERK and negatively regulates NF‐κB, we investigated whether there existed any crosstalk between the ERK and the NF‐κB pathways. NF‐κB‐dependent reporter assay, visualization of the nuclear translocation of NF‐κB‐p65 subunit and determination of the cellular levels of I‐κB, the inhibitor of NF‐κB, revealed that SAC inhibited TNF‐α‐induced NF‐κB activation, and PD98059 treatment reversed this effect. These results collectively suggest that SAC inhibits TNF‐α‐induced inflammation in HaCaT cells via a combined effect entailing the inhibition of the p38 and the JNK pathways and NF‐κB pathway via the sustained activation of ERK.     

Baicalein inhibits matrix metalloproteinase 1 expression via activation of TRPV1‐Ca‐ERK pathway in ultraviolet B–irradiated human dermal fibroblasts

Increased matrix metalloproteinase 1 (MMP‐1) expression is a feature of photo‐aged skin. We investigated the effects of baicalein and sulphoraphane on ultraviolet B (UVB) irradiation–induced MMP‐1 expression and apoptosis using human dermal fibroblasts. UVB irradiation not only increased MMP‐1 expression, but also caused apoptosis. Both baicalein and sulphoraphane protected cells from UVB irradiation–induced apoptosis, but only baicalein inhibited MMP‐1 expression. UVB irradiation activated 12‐lipoxygenase, and its product, 12‐hydroxyeicosatetraenoic acid, activated TRPV1 channels. The resulting UVB irradiation–induced Ca²⁺ increase was blocked by the 12‐lipoxygenase inhibitor baicalein and the TRPV1 blocker capsazepine, but not by the Nrf2 inducer sulphoraphane. UVB irradiation also increased ROS generation and decreased Nrf2 protein levels. UVB irradiation–induced MMP‐1 expression was blocked by the Ca²⁺ chelator BAPTA, by capsazepine and by TRPV1 silencing. However, induction was unaffected by the antioxidant N‐acetylcysteine. ERK phosphorylation and JNK phosphorylation were induced by UVB irradiation, but only ERK phosphorylation was Ca²⁺ sensitive. Increased MMP‐1 expression was blocked by PD98059, but not by SP600125. Thus, increased MMP‐1 expression is mediated by increased cytosolic Ca²⁺ and ERK phosphorylation. UVB irradiation–induced ROS generation is also Ca²⁺ sensitive, and UVB irradiation–induced apoptosis is caused by increased ROS. Thus, baicalein, by blocking the UVB irradiation–induced cytosolic Ca²⁺ increase, protects cells from UVB irradiation–induced MMP‐1 expression and apoptosis. In contrast, sulphoraphane, by decreasing cellular ROS, protects cells from only UVB‐induced apoptosis. Thus, targeting 12‐lipoxygenase may provide a therapeutic approach to improving the health of photo‐aged human skin.     

Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes

Please cite this paper as: Activation of toll‐like receptors 2, 3 or 5 induces matrix metalloproteinase‐1 and ‐9 expression with the involvement of MAPKs and NF‐κB in human epidermal keratinocytes. Experimental Dermatology 2010; 19 : e44–e49. Abstract: Toll‐like receptors (TLRs) on epidermal keratinocytes are the first line of defense against microbe invasion, and matrix metalloproteases (MMPs) regulate inflammation, cell migration and wound healing. In this study, we demonstrate that the mRNA and protein expressions of MMP‐1 and MMP‐9 in human epidermal keratinocytes are induced by ligands for TLR2, TLR3 and TLR5 [Pam3CSK4, Poly(I:C) and flagellin, respectively] in a dose‐dependent manner. We also found that the ligands for TLR2, TLR3 and TLR5 activate the MAP kinases, JNK and p38 MAPK, but not ERK1/2. Furthermore, treatment with the ligands for TLR2, TLR3 and TLR5 also induced the degradation of IκB‐α and activated the nuclear translocation of NF‐κB. MMP‐1 induction by the ligands for TLR2, TLR3 and TLR5 was inhibited by pretreatment with BAY11‐7082 (NF‐κB inhibitor) or SP600125 (JNK inhibitor), whereas MMP‐9 expression was inhibited by pretreatment with BAY11‐7082, SP600125 or SB203580. These findings demonstrate that the activation of TLR2, TLR3 or TLR5 induces the expression of MMP‐1 and MMP‐9 in human epidermal keratinocytes. In addition, NF‐κB or JNK mediated the MMP‐1 expression induced by TLR2, TLR3 and TLR5, whereas NF‐κB, JNK or p38 MAPK mediated the MMP‐9 expression induced by TLR2, TLR3 and TLR5.     

Azelaic acid reduced senescence‐like phenotype in photo‐irradiated human dermal fibroblasts: possible implication of PPARγ

Azelaic acid (AzA) has been used for the treatment for inflammatory skin diseases, such as acne and rosacea. Interestingly, an improvement in skin texture has been observed after long‐time treatment with AzA. We previously unrevealed that anti‐inflammatory activity of AzA involves a specific activation of PPARγ, a nuclear receptor that plays a relevant role in inflammation and even in ageing processes. As rosacea has been considered as a photo‐aggravated disease, we investigated the ability of AzA to counteract stress‐induced premature cell senescence (SIPS). We employed a SIPS model based on single exposure of human dermal fibroblasts (HDFs) to UVA and 8‐methoxypsoralen (PUVA), previously reported to activate a senescence‐like phenotype, including long‐term growth arrest, flattened morphology and increased synthesis of matrix metalloproteinases (MMPs) and senescence‐associated β‐galactosidase (SA‐β‐gal). We found that PUVA‐treated HDFs grown in the presence of AzA maintained their morphology and reduced MMP‐1 release and SA‐β‐galactosidase‐positive cells. Moreover, AzA induced a reduction in ROS generation, an up‐modulation of antioxidant enzymes and a decrease in cell membrane lipid damages in PUVA‐treated HDFs. Further evidences of AzA anti‐senescence effect were repression of p53 and p21, increase in type I pro‐collagen and abrogation of the enhanced expression of growth factors, such as HGF and SCF. Interestingly, PUVA‐SIPS showed a decreased activation of PPARγ and AzA counteracted this effect, suggesting that AzA effect involves PPARγ modulation. All together these data showed that AzA interferes with PUVA‐induced senescence‐like phenotype and its ability to activate PPAR‐γ provides relevant insights into the anti‐senescence mechanism.     

Z‐ligustilide ameliorated ultraviolet B‐induced oxidative stress and inflammatory cytokine production in human keratinocytes through upregulation of Nrf2/HO‐1 and suppression of NF‐κB pathway

Ultraviolet B (UVB), a harmful environmental factor, is responsible for a variety of skin disorders including skin inflammation through reactive oxygen species (ROS) and inflammatory mediator production. Here, we investigated the effect of Z‐ligustilide (Z‐lig), an active ingredient isolated from the medicinal plants Cnidium officinale and Angelica acutiloba, on UVB‐induced ROS generation and inflammatory mediator production in normal human epidermal keratinocytes (NHEKs) as well as its underlying mechanisms. Z‐lig significantly rescued UVB‐induced NHEKs damage in a dosage‐dependent manner. Pretreatment of NHEKs with Z‐lig inhibited UVB‐induced ROS production in NHEKs. Both silencing the nuclear factor E2‐related factor 2 (Nrf2) and the supplement of tin protoporphyrin IX (SnPP), a haeme oxygenase‐1 (HO‐1) inhibitor, cancelled the inhibitory effect of Z‐lig on UVB‐induced ROS upregulation in NHEKs. Moreover, pretreatment of NHEKs with Z‐lig reduced UVB‐induced nuclear factor kappa B (NF‐κB)‐dependent inflammatory mediators (IL‐6, IL‐8 and MCP‐1) production at both mRNA and protein level. In the presence of Z‐lig, UVB‐induced NF‐κB subunit p65 nuclear translocation was abolished, and the IκBα degradation was suppressed. Taken together, these findings suggest that Z‐lig can suppress UVB‐induced ROS generation through Nrf2/HO‐1 upregulation and inflammation by suppressing the NF‐κB pathway, suggesting that Z‐lig may be beneficial in protecting skin from UVB exposure.     

Protective effect of pomegranate-derived products on UVB-mediated damage in human reconstituted skin

Abstract: Solar ultraviolet (UV) radiation, particularly its UVB (290–320 nm) component, is the primary cause of many adverse biological effects including photoageing and skin cancer. UVB radiation causes DNA damage, protein oxidation and induces matrix metalloproteinases (MMPs). Photochemoprevention via the use of botanical antioxidants in affording protection to human skin against UVB damage is receiving increasing attention. Pomegranate, from the tree Punica granatum, contains anthocyanins and hydrolysable tannins and possesses strong antioxidant and anti‐tumor‐promoting properties. In this study, we determined the effect of pomegranate‐derived products – POMx juice, POMx extract and pomegranate oil (POMo) – against UVB‐mediated damage using reconstituted human skin (EpiDerm^TM FT‐200). EpiDerm was treated with POMx juice (1–2 μl/0.1 ml/well), POMx extract (5–10 μg/0.1 ml/well) and POMo (1–2 μl/0.1 ml/well) for 1 h prior to UVB (60 mJ/cm²) irradiation and was harvested 12 h post‐UVB to assess protein oxidation, markers of DNA damage and photoageing by Western blot analysis and immunohistochemistry. Pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) cyclobutane pyrimidine dimers (CPD), (ii) 8‐dihydro‐2′‐deoxyguanosine (8‐OHdG), (iii) protein oxidation and (iv) proliferating cell nuclear antigen (PCNA) protein expression. We also found that pretreatment of Epiderm with pomegranate‐derived products resulted in inhibition of UVB‐induced (i) collagenase (MMP‐1), (ii) gelatinase (MMP‐2, MMP‐9), (iii) stromelysin (MMP‐3), (iv) marilysin (MMP‐7), (v) elastase (MMP‐12) and (vi) tropoelastin. Gelatin zymography revealed that pomegranate‐derived products inhibited UVB‐induced MMP‐2 and MMP‐9 activities. Pomegranate‐derived products also caused a decrease in UVB‐induced protein expression of c‐Fos and phosphorylation of c‐Jun. Collectively, these results suggest that all three pomegranate‐derived products may be useful against UVB‐induced damage to human skin.     

Loss of INK4a/Arf gene enhances ultraviolet radiation‐induced cutaneous tumor development

The CDKN2A locus encodes for tumor suppressor genes p16^INK4a and p14^Arf which are frequently inactivated in human skin tumors. The purpose of this study was to determine the relationship between loss of INK4a/Arf activity and inflammation in the development of ultraviolet (UV) radiation‐induced skin tumors. Panels of INK4a/Arf^‐/? mice and wild‐type (WT) mice were treated with a single dose of UVB (200 mJ/cm²). For long‐term studies, these mice were irradiated with UVB (200 mJ/cm²) three times weekly for 30 weeks. At the end of the experiment, tissues were harvested from mice and assayed for inflammatory biomarkers and cytokines. A single dose of UVB resulted in a significant increase in reactive oxygen species (ROS) and 8‐dihydroxyguanosine (8‐oxo‐dG) lesions in INK4a/Arf^?/? mice compared to WT mice. When subjected to chronic UVB, we found that 100% of INK4a/Arf^?/‐ mice had tumors, whereas there were no tumors in WT controls after 24 weeks of UVB exposure. The increase in tumor development correlated with a significant increase in nuclear factor (NF)‐κB, cyclooxygenase‐2 (COX‐2), prostaglandin E₂ (PGE₂) and its receptors both in UVB‐exposed skin and in the tumors. A significant increase was seen in inflammatory cytokines in skin samples of INK4a/Arf^‐/‐ mice following treatment with chronic UVB radiation. Furthermore, significantly more CD11b⁺Gr1⁺ myeloid cells were present in UVB‐exposed INK4a/Arf^‐/‐ mice compared to WT mice. Our data indicate that by targeting UVB‐induced inflammation, it may be possible to prevent UVB‐induced skin tumors in individuals that carry CDKN2A mutation.     

Serum amyloid A1 secreted from UV‐irradiated keratinocytes induces matrix metalloproteinase‐1 in fibroblasts through toll‐like receptor 4

Ultraviolet (UV) irradiation on skin triggers photoageing‐related phenotypes such as formation of wrinkles. UV ray upregulates matrix metalloproteinase‐1 (MMP‐1), which in turn degrades extracellular matrix proteins, mostly collagens. Serum amyloid A1 (SAA1) is an acute‐phase protein of which plasma concentration increases in response to inflammation. Although the expression of SAA1 in the skin was reported, its function in the skin is yet to be studied. In this research, we found that the expression of SAA1 was increased in acute UV‐irradiated buttock skin and photoaged forearm skin in vivo. UV irradiation also increased SAA1 in normal human epidermal keratinocytes (NHEK), and treatment of recombinant human SAA1 (rhSAA1) induced MMP‐1 in normal human dermal fibroblasts (NHDF) but not in NHEK. Next, we demonstrated that NHDF treated with UV‐irradiated keratinocyte‐conditioned media showed the increased MMP‐1 expression; however, this increase of MMP‐1 in NHDF was inhibited by knockdown of SAA1 in NHEK. In addition, knockdown of Toll‐like receptor 4 (TLR4) inhibited rhSAA1‐induced MMP‐1 expression in NHDF. Taken together, our data showed that UV‐induced SAA1 production in NHEK, and this secreted SAA1 induced MMP‐1 expression in NHDF in a paracrine manner through TLR4 signalling pathway. Therefore, our results suggest that SAA1 can be a potential mediator for UV‐induced MMP‐1 expression in human skin.