Ultraviolet A-induced production of matrix metalloproteinase-1 is mediated by macrophage migration inhibitory factor (MIF) in human dermal fibroblasts

Matrix metalloproteinases (MMPs) are thought to be responsible for dermal photoaging in human skin. In the present study, we evaluated the involvement of macrophage migration inhibitory factor (MIF) in MMP-1 expression under ultraviolet A (UVA) irradiation in cultured human dermal fibroblasts. UVA (20 J/cm(2)) up-regulates MIF production, and UVA-induced MMP-1 mRNA production is inhibited by an anti-MIF antibody. MIF (100 ng/ml) was shown to induce MMP-1 in cultured human dermal fibroblasts. We found that MIF (100 ng/ml) enhanced MMP-1 activity in cultured fibroblasts assessed by zymography. Moreover, we observed that fibroblasts obtained from MIF-deficient mice were much less sensitive to UVA regarding MMP-13 expression than those from wild-type BALB/c mice. Furthermore, after UVA irradiation (10 J/cm(2)), dermal fibroblasts of MIF-deficient mice produced significantly decreased levels of MMP-13 compared with fibroblasts of wild-type mice. Next we investigated the signal transduction pathway of MIF. The up-regulation of MMP-1 mRNA by MIF stimulation was found to be inhibited by a PKC inhibitor (GF109203X), a Src-family tyrosine kinase inhibitor (herbimycin A), a tyrosine kinase inhibitor (genistein), a PKA inhibitor (H89), a MEK inhibitor (PD98089), and a JNK inhibitor (SP600125). In contrast, the p38 inhibitor (SB203580) was found to have little effect on expression of MMP-1 mRNA. We found that PKC-pan, PKC alpha/beta II, PKC delta (Thr505), PKC delta (Ser(643)), Raf, and MAPK were phosphorylated by MIF. Moreover, we demonstrated that phosphorylation of PKC alpha/beta II and MAPK in response to MIF was suppressed by genistein, and herbimycin A as well as by transfection of the plasmid of C-terminal Src kinase. The DNA binding activity of AP-1 was significantly up-regulated 2 h after MIF stimulation. Taken together, these results suggest that MIF is involved in the up-regulation of UVA-induced MMP-1 in dermal fibroblasts through PKC-, PKA-, Src family tyrosine kinase-, MAPK-, c-Jun-, and AP-1-dependent pathways.     

Alpha-lipoic acid induces apoptosis in hepatoma cells via the PTEN/Akt pathway

We report here that alpha-lipoic acid (alpha-LA), a naturally-occurring antioxidant, scavenges reactive oxygen species (ROS) followed by an increase in apoptosis of human hepatoma cells. Apoptosis induced by alpha-LA was dependent upon the activation of the caspase cascade and the mitochondrial death pathway. alpha-LA induced increases in caspase-9 and caspase-3 but had no significant effect on caspase-8 activity. Apoptosis induced by alpha-LA was found to be mediated through the tensin homologue deleted on chromosome 10 (PTEN)/Akt pathway. Prior to cell apoptosis, PTEN was activated and its downstream target Akt was inhibited. Our findings indicate that increasing ROS scavenging could be a therapeutic strategy to treat cancer.     

The effect of cilostazol on the expression of matrix metalloproteinase-1 and type I procollagen in ultraviolet-irradiated human dermal fibroblasts

AimCilostazol is a selective inhibitor of type III phosphodiesterase that inhibits platelet aggregation. Cilostazol is a useful vasodilator, antithrombotic, and cardiotonic agent. Ultraviolet B (UVB) irradiation increases the production of matrix metalloproteinase-1 (MMP-1) during skin photoaging. The UVB-induced increase of MMP-1 results in connective tissue damage, and the skin becomes wrinkled and aged. Here, we investigated the capacity of cilostazol to inhibit MMP-1 expression in UVB-irradiated human dermal fibroblasts.Main methodsCultured human dermal fibroblasts were irradiated with UVB, followed by the addition of cilostazol to the culture medium.Key findingsPost-treatment with cilostazol attenuated UVB-induced production of MMP-1 and prevented the reduction of type I procollagen. Cilostazol inhibited UVB irradiation-induced phosphorylation of the mitogen-activated protein kinase (MAPK) signaling molecules Jun-N-terminal kinase (JNK) and p38 kinase, as well as activator protein-1 (AP-1) in dermal fibroblasts.SignificanceOverall, these results demonstrate that cilostazol regulates UVB-induced MMP-1 expression and type I procollagen synthesis by inhibiting MAPK signaling and AP-1 activity. Therefore, we suggest that cilostazol may be useful for the prevention and treatment of skin photodamage caused by UVB-irradiation.     

Transcriptional regulation of matrix metalloproteinase-1 and collagen 1A2 explains the anti-fibrotic effect exerted by proteasome inhibition in human dermal fibroblasts

Introduction  

Extracellular matrix (ECM) turnover is controlled by the synthetic rate of matrix proteins, including type I collagen, and their enzymatic degradation by matrix metalloproteinases (MMPs). Fibrosis is characterized by an unbalanced accumulation of ECM leading to organ dysfunction as observed in systemic sclerosis. We previously reported that proteasome inhibition (PI) in vitro decreases type I collagen and enhances MMP-1 production by human fibroblasts, thus favoring an antifibrotic fibroblast phenotype. These effects were dominant over the pro-fibrotic phenotype induced by transforming growth factor (TGF)-β. Here we investigate the molecular events responsible for the anti-fibrotic phenotype induced in fibroblasts by the proteasome inhibitor bortezomib.     

Group IB secretory phospholipase A2 promotes matrix metalloproteinase-2-mediated cell migration via the phosphatidylinositol 3-kinase and Akt pathway

Secretory phospholipase A(2) (sPLA(2)), abundantly expressed in various cells including fibroblasts, is able to promote proliferation and migration. Degradation of collagenous extracellular matrix by matrix metalloproteinase (MMP) plays a role in the pathogenesis of various destructive disorders, such as rheumatoid arthritis, tumor invasion, and metastasis. Here we show that group IB PLA(2) increased pro-MMP-2 activation in NIH3T3 fibroblasts. MMP-2 activity was stimulated by group IB PLA(2) in a dose- and time-dependent manner. Consistent with MMP-2 activation, sPLA(2) decreased expression of type IV collagen. These effects are due to the reduction of tissue inhibitor of metalloproteinase-2 (TIMP-2) and the activation of the membrane type1-MMP (MT1-MMP). The decrease of TIMP-2 levels in conditioned media and the increase of MT1-MMP levels in plasma membrane were observed. In addition, treatment of cells with decanoyl Arg-Val-Lys-Arg-chloromethyl ketone, an inhibitor of pro-MT1-MMP, suppressed sPLA(2)-mediated MMP-2 activation, whereas treatment with bafilomycin A1, an inhibitor of H(+)-ATPase, sustained MMP-2 activation by sPLA(2). The involvement of phosphatidylinositol 3-kinase (PI3K) and Akt in the regulation of MMP-2 activity was further suggested by the findings that PI3K and Akt were phosphorylated by sPLA(2). Expression of p85alpha and Akt mutants, or pretreatment of cells with LY294002, a PI3K inhibitor, attenuated sPLA(2)-induced MMP-2 activation and migration. Taken together, these results suggest that sPLA(2) increases the pro-MMP-2 activation and migration of fibroblasts via the PI3K and Akt-dependent pathway. Because MMP-2 is an important factor directly involved in the control of cell migration and the turnover of extracellular matrix, our study may provide a mechanism for sPLA(2)-promoted fibroblasts migration.     

Thioredoxin secreted upon ultraviolet A irradiation modulates activities of matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-2 in human dermal fibroblasts

Regulation of the balance of matrix metalloproteinase-2 (MMP-2) and its tissue inhibitor (TIMP-2) by thioredoxin (Trx) was investigated in human dermal fibroblasts. Expression and secretion of Trx and Trx reductase 1 (TR1) was increased after ultraviolet (UV) A irradiation. A significant increase in proMMP-2 activity and a decrease of TIMP-2 activity in supernatants of UVA-irradiated fibroblasts were observed in gelatin and reverse zymography compared to non-irradiated fibroblasts. Removal of Trx or TR1 by immunoprecipitation diminished these changes in proMMP-2 activity. Incubation with 5, 5'-dithio-bis-2-nitrobenzoic acid (DTNB) also suppressed these changes. Incubation with recombinant Trx or TR decreased TIMP-2 activity and increased MMP-2 activity. UVA-irradiated fibroblasts, transiently transfected with a dominant-negative mutant or wild-type Trx, showed down- or upregulation of proMMP-2 activities, respectively, without significant change of protein amount. In conclusion, thioredoxin secreted by UVA irradiation is involved in the regulation of MMP-2 and TIMP-2 activities through its redox activity in human dermal fibroblasts.     

Interleukin-1 stimulates cytokines, prostaglandin E2 and matrix metalloproteinase-1 production via activation of MAPK/AP-1 and NF-kappaB in human gingival fibroblasts

Interleukin-1 (IL-1) plays a crucial role in the immunopathological responses involved with tissue destruction in chronic inflammatory diseases, such as periodontal disease, as it stimulates host cells including fibroblasts to produce various inflammatory mediators and catabolic factors. We comprehensively investigated the involvement of mitogen-activated protein kinases (MAPKs)/activator protein-1 (AP-1) and IkappaB kinases (IKKs)/IkappaBs/nuclear factor-kappaB (NF-kappaB) in IL-1beta-stimulated IL-6, IL-8, prostaglandin E(2) (PGE(2)) and matrix metalloproteinase-1 (MMP-1) production by human gingival fibroblasts (HGF). Three MAPKs, extracellular signal-regulated kinase (ERK), p38 MAPK and c-Jun N-terminal kinase (JNK), which were simultaneously activated by IL-1beta, mediated subsequent c-fos and c-jun mRNA expression and DNA binding of AP-1 at different magnitudes. IKKalpha/beta/IkappaB-alpha/NF-kappaB was also involved in the IL-1 signaling cascade. Further, IL-1beta stimulated HGF to produce IL-6, IL-8, PGE(2) and MMP-1 via activation of the 3 MAPKs and NF-kappaB, as inhibitors of each MAPK and NF-kappaB significantly suppressed the production of IL-1beta-stimulated factors, though these pathways might also play distinct roles in IL-1beta activities. Our results strongly suggest that the MAPKs/AP-1 and IKK/IkappaB/NF-kappaB cascades cooperatively mediate the IL-1beta-stimulated synthesis of IL-6, IL-8, PGE(2) and MMP-1 in HGF.     

Overexpression of UBR5 promotes tumor growth in gallbladder cancer via PTEN/PI3K/Akt signal pathway

As a key regulator of the ubiquitin-proteasome system, ubiquitin protein ligase E3 component N-recognin 5 (UBR5) plays an important role in various cancers. In this study, our results showed for the first time that UBR5 was overexpressed in gallbladder cancer (GBC) tumor tissues. UBR5 overexpression was significantly associated with tumor size, histological and tumor differentiation. UBR5 overexpression was also associated with poor prognosis in patients with GBC. The knockdown of UBR5 remarkably inhibited the cell proliferation and colony formation of GBC-Shandong (SD) cells in vitro and in vivo. UBR5 potentially increases the level of protein kinase B phosphorylation via the degradation of phosphatase and tensin homolog, which contributes to tumor growth in GBC. UBR5 may be an important biomarker for predicting the prognosis of patients with GBC.     

Fli-1 inhibits collagen type I production in dermal fibroblasts via an Sp1-dependent pathway

Fibrosis is characterized by the excessive deposition of extracellular matrix (ECM), especially collagen. Because Ets factors are implicated in physiological and pathological ECM remodeling, the aim of this study was to investigate the role of Ets factors in collagen production. We demonstrate that the expression of collagenous proteins and collagen alpha2(I) (COL1A2) mRNA was inhibited following stable transfection of Fli-1 in dermal fibroblasts. Subsequent analysis of the COL1A2 promoter identified a critical Ets binding site that mediates Fli-1 inhibition. In contrast, Ets-1 stimulates COL1A2 promoter activity. In vitro binding assays demonstrate that both Fli-1 and Ets-1 form DNA-protein complexes with sequences present in COL1A2 promoter. Furthermore, Fli-1 binding to the COL1A2 is enhanced via Sp1-dependent interaction. Studies using Fli-1 dominant interference and DNA binding mutants indicate that Fli-1 inhibition is mediated by both direct (DNA binding) and indirect (via protein-protein interaction) mechanisms and that Sp1 is an important mediator of the Fli-1 function. Furthermore, experiments using the Gal4 system in the context of different cell types as well as experiments with the COL1A2 promoter in different cell lines demonstrate that the direction and magnitude of the effect of Fli-1 is promoter- and cell context-specific. We propose that Fli-1 inhibits COL1A2 promoter activity by competition with Ets-1. In addition, we postulate that another factor (co-repressor) may be required for maximal inhibition after recruitment to the Fli-1-Sp1 complex. We conclude that the ratio of Fli-1 to Ets-1 and the presence of co-regulatory proteins ultimately control ECM production in fibroblasts.     

Wnt5a promotes adhesion of human dermal fibroblasts by triggering a phosphatidylinositol-3 kinase/Akt signal

Frizzled-3 (Fzd3), highly expressed in both the central nervous system (CNS) and skin, plays essential roles in axonal growth and guidance during the CNS development and may be involved in maintenance of skin integrity, although its ligand remains undetermined. In this study, we demonstrate that Wnt5a specifically binds to Fzd3 in vitro and triggers phosphorylation of Akt mediated by phosphatidylinositol-3 kinase (PI3K), but not that of ERK or protein kinase C, in human primary-cultured dermal fibroblasts. We have further found that such Wnt5a/Fzd3-triggered activation of the PI3K/Akt signal promotes integrin-mediated adhesion of human dermal fibroblasts to collagen I-coated dishes. Based on another finding that Wnt5a/Fzd3-triggered activation of the PI3K/Akt signal was blocked by an excess amount of a recombinant Fzd3-cysteine-rich domain (CRD), but not by that of a recombinant Fzd6-CRD, it is concluded that Wnt5a is a natural ligand of Fzd3 that triggers the PI3K/Akt signal and promotes adhesion of human dermal fibroblasts.     

High glucose enhances MMP-2 production in adventitial fibroblasts via Akt1-dependent NF-kappaB pathway

To understand the role of adventitial fibroblasts (AF) in diabetic vascular diseases, the importance of high glucose (HG, 25mM) on matrix metalloproteinase-2 (MMP-2) production in AF was determined. HG enhanced mRNA, protein and gelatinolytic activity of MMP-2. The enhanced MMP-2 activity was significantly attenuated not only by a PI3K inhibitor but also by an Akt inhibitor. These HG-induced MMP-2 responses were markedly reduced in Akt1-deficient (1KO) cells. The diminished HG-induced MMP-2 responses were completely restored by re-expression of Akt1. Both the reporter activity and electrophoretic mobility shift assay for activator protein-1 and nuclear factor-kappa B (NF-kappaB) were enhanced by HG, but NF-kappaB were not increased in 1KO cells. Furthermore, HG-induced MMP-2 responses were markedly suppressed by NF-kappaB decoy oligodeoxynucleotides. Based on these results, it is suggested that HG augments MMP-2 production via PI3K/Akt1/NF-kappaB pathway.