Chi-Chin Sun

Sphingosine‐1‐phosphate induces COX‐2 expression via PI3K/Akt and p42/p44 MAPK pathways in rat vascular smooth muscle cells

Sphingosine 1‐phosphate (S1P) has been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. S1P increases the expression of several proteins including COX‐2 in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating COX‐2 expression by S1P in VSMCs remain unclear. Western blotting and RT‐PCR analyses showed that S1P induced the expression of COX‐2 mRNA and protein in a time‐ and concentration‐dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and PI3K (wortmannin), and transfection with dominant negative mutants of p42/p44 mitogen‐activated protein kinases (ERK2) or Akt. These results suggested that both p42/p44 MAPK and PI3K/Akt pathways participated in COX‐2 expression induced by S1P in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt, which was attenuated by U0126, LY294002, or wortmannin, respectively. Furthermore, this up‐regulation of COX‐2 mRNA and protein was blocked by a selective NF‐κB inhibitor helenalin. Consistently, S1P‐stimulated translocation of NF‐κB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P‐stimulated activation of NF‐κB promoter activity was blocked by phosphatidylinositol 3‐kinase (PI3K) inhibitor LY294002 and helenalin, but not by U0126, suggesting that involvement of PI3K/Akt in the activation of NF‐κB. COX‐2 promoter assay showed that S1P induced COX‐2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF‐κB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF‐κB pathways was essential for S1P‐induced COX‐2 gene expression. Understanding the mechanisms involved in S1P‐induced COX‐2 expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis. J. Cell. Physiol.

Ocular Biocompatibility of Carbodiimide Cross-Linked Hyaluronic Acid Hydrogels for Cell Sheet Delivery Carriers

Due to its innocuous nature, hyaluronic acid (HA) is one of the most commonly used biopolymers for ophthalmic applications. We recently developed a cell sheet delivery system using carbodiimide cross-linked HA carriers. Chemical cross-linking provides an improvement in stability of polymer gels, but probably causes toxic side-effects. The aim of this study was to investigate the ocular biocompatibility of HA hydrogels cross-linked by 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC). HA discs without cross-linking and glutaraldehyde (GTA) cross-linked HA samples were used for comparison. The disc implants were inserted in the anterior chamber of rabbit eyes for 24 weeks and characterized by slit-lamp biomicroscopy, histology and scanning electron microscopy. The ophthalmic parameters obtained from biomicroscopic examinations were also scored to provide a quantitative grading system. Results of this study showed that the HA discs cross-linked with EDC had better ocular biocompatibility than those with GTA. The continued residence of GTA cross-linked HA implants in the intraocular cavity elicited severe tissue responses and significant foreign body reactions, whereas no adverse inflammatory reaction was observed after contact with non-cross-linked HA or EDC cross-linked HA samples. It is concluded that the cross-linking agent type gives influence on ocular biocompatibility of cell carriers and the EDC-HA hydrogel is an ideal candidate for use as an implantable material in cell sheet delivery applications.

Persistence of Transplanted Oral Mucosal Epithelial Cells in Human Cornea

PURPOSE To determine the expression of differentiation and progenitor cell markers in corneal tissues that previously underwent autologous cultivated oral mucosal epithelial transplantation (COMET). METHODS Four eyes from three alkaline-injured patients and one thermally injured patient underwent COMET to promote re-epithelialization or corneal reconstruction. Between 10 and 22 months (mean, 14.2 +/- 5.5 months [SD]) after COMET, the corneal tissues were obtained after penetrating keratoplasty (n = 1) or autologous limbal transplantation (n = 3). Immunoconfocal microscopy for keratin (K)3, -12, -4, -13, and -8; connexin (Cx)43; MUC5AC; laminin-5; pan-p63; ABCG2; and p75 was performed in those specimens as well as in the oral mucosa and cultivated oral mucosal epithelial cells (OMECs). RESULTS All four specimens were unanimously positive for K3, -4, and -13 but negative for K8 and MUC5AC, suggesting that the keratinocytes were oral mucosa-derived. However, peripheral K12 staining was positive only in patient 2, suggesting a mixed oral and corneal epithelium in that case. Cx43 staining in the basal epithelium was negative in patients 1, 2, and 3, but was positive in patient 4. Small, compact keratinocytes in the basal epithelium preferentially expressed pan-p63, ABCG2, and p75. Although the staining of pan-p63 and ABCG2 tended to be more than one layer, signal for p75 was consistently localized only to the basal layer. CONCLUSIONS The study demonstrated the persistence of transplanted OMECs in human corneas. In addition, small, compact cells in the basal epithelium preferentially expressed the keratinocyte stem/progenitor cell markers, which may be indicative of the engraftment of the progenitor cells after transplantation.

IL‐1β induces MMP‐9 expression via a Ca2+‐dependent CaMKII/JNK/c‐JUN cascade in rat brain astrocytes

Interleukin (IL)‐1β has been shown to induce matrix metalloproteinase (MMP)‐9 expression through mitogen‐activated protein kinases, including JNK, in rat brain astrocyte‐1 (RBA‐1) cells. However, little is known about whether JNK activated by Ca2+‐dependent CaMKII is associated with MMP‐9 expression induced by IL‐1β. Here, we report that the Ca2+/CaMKII/JNK/c‐Jun participates in the MMP‐9 expression induced by IL‐1β. Zymographic, Western blotting, and RT‐PCR analyses showed that IL‐1β‐induced expression of MMP‐9 mRNA and protein was attenuated by Ca2+ chelator (BAPTA), and the inhibitors of ER Ca2+‐ATPase (thapsigargin), CaMKII (KN‐62), and JNK1/2 (SP600125). IL‐1β also stimulated phosphorylation of CaMKII and JNK1/2, and increase in intracellular Ca2+ ([Ca2+]i), which were inhibited by pretreatment with BAPTA, thapsigargin (TG), KN‐62, or SP600125. Furthermore, the upregulation of MMP‐9 protein was blocked by transfection with c‐Jun or CaMKII short hairpin RNA (shRNA). We further confirmed that IL‐1β stimulated c‐Jun associated with AP‐1‐binding sites within MMP‐9 promoter (−87 to −80 bp and −511 to −497 bp) by immunoprecipitation and chromatin immunoprecipitation (ChIP)‐PCR assays. The activation and recruitment of c‐Jun to MMP‐9 promoter were inhibited by pretreatment with BAPTA, TG, KN‐62, or SP600125. Moreover, IL‐1β‐induced MMP‐9 gene transcription by AP‐1 was confirmed by transfection with a MMP‐9 promoter‐luciferase reporter plasmid with a distal AP‐1‐binding site (−511 to −497 bp) adjacent to an Ets‐binding site‐mutation (mt‐AP1/Ets‐MMP‐9). These results demonstrated that in RBA‐1 cells, JNK/c‐Jun activation was mediated through a Ca2+‐dependent CaMKII pathway that promoted transcription factor c‐Jun/AP‐1 recruitment and eventually led to increase in MMP‐9 expression by IL‐1β.

PKC-δ/c-Src-mediated EGF receptor transactivation regulates thrombin-induced COX-2 expression and PGE2 production in rat vascular smooth muscle cells

The thrombin/proteinase-activated receptors (PARs) have been shown to regulate smooth muscle cell proliferation, migration, and vascular maturation. Thrombin up-regulates expression of several proteins including cyclooxygenase (COX)-2 in vascular smooth muscle cells (VSMCs) and contributes to vascular diseases. However, the mechanisms underlying thrombin-regulated COX-2 expression in VSMCs remain unclear. Western blotting, RT-PCR, and EIA kit analyses showed that thrombin induced the expression of COX-2 mRNA and protein and PGE(2) release in a time-dependent manner, which was attenuated by inhibitors of PKC (GF109203X and rottlerin), c-Src (PP1), EGF receptor (EGFR; AG1478) and MEK1/2 (U0126), or transfection with dominant negative mutants of PKC-delta, c-Src or extracellular regulated kinase (ERK) and ERK1 short hairpin RNA interference (shRNA). These results suggest that transactivation of EGFR participates in COX-2 expression induced by thrombin in VSMCs. Accordingly, thrombin stimulated phosphorylation of ERK1/2 which was attenuated by GF109203X, rottlerin, PP1, GM6001, CRM197, AG1478, or U0126, respectively. Furthermore, this up-regulation of COX-2 mRNA and protein was blocked by selective inhibitors of AP-1 and NF-kappaB, curcumin and helenalin, respectively. Moreover, thrombin-stimulated activation of NF-kappaB, AP-1, and COX-2 promoter activity was blocked by the inhibitors of c-Src, PKC, EGFR, MEK1/2, AP-1 and NF-kappaB, suggesting that thrombin induces COX-2 promoter activity mediated through PKC(delta)/c-Src-dependent EGFR transactivation, MEK-ERK1/2, AP-1, and NF-kappaB. These results demonstrate that in VSMCs, activation of ERK1/2, AP-1 and NF-kappaB pathways was essential for thrombin-induced COX-2 gene expression. Understanding the regulation of COX-2 expression and PGE(2) release by thrombin/PARs system on VSMCs may provide potential therapeutic targets of vascular inflammatory disorders including arteriosclerosis.

IL-1β induces urokinse-plasminogen activator expression and cell migration through PKCα, JNK1/2, and NF-κB in A549 cells

Breakdown of the extracellular matrix (ECM) is accomplished by the concerted action of several proteases, including the urokinase plasminogen‐activator (uPA) system and matrix metalloproteinases (MMPs), which is crucial for cancer invasion and metastasis. Several reports have shown that the levels of IL‐1β and MMPs in plasma of the patients with lung cancer are significantly elevated and link to the invasion of tumor cells. Therefore, we investigated whether IL‐1β‐induced expression of uPA participated in lung cancer progression. In this study, IL‐1β significantly induced uPA expression and activity via PKCα‐dependent JNK1/2 and NIK cascades, linking to IKKα/β activation, p65 translocation and transcription activity, using pharmacological inhibitors and transfection with dominant negative mutants and siRNAs. IL‐1β‐induced uPA protein and mRNA expression in a time‐ and concentration‐dependent manner, which was inhibited by pretreatment with the inhibitors of JNK1/2 (SP600125), PKC (Ro31‐8220, Gö6976), or NF‐κB (helenalin), and transfection with dominant negative mutants of PKCα, NIK, and IKKβ, and siRNAs of JNK1/2 and p65. IL‐1β stimulated PKCα translocation to plasma membrane leading to phosphorylation of JNK1/2, which was attenuated by PKC inhibitors and transfection with shRNAs of JNK1/2, but not by helenalin. In addition, IL‐1β stimulated p65 phosphorylation and translocation into nucleus concomitant with IκBα phosphorylation and IκBα degradation, which was mediated via activation of PKCα‐dependent JNK1/2–NIK/IKKβ cascade. These results demonstrated that in A549 cells, activation of p50/p65 heterodimer through sequential activation of PKCα–JNK–NIK–IKKβ–NF‐κB was required for IL‐1β‐induced uPA expression associated with migration of tumor cells. J. Cell. Physiol. 219: 183–193, 2009.

Oxidized low-density lipoprotein induces matrix metalloproteinase-9 expression via a p42/p44 and JNK-dependent AP-1 pathway in brain astrocytes

Upregulation of matrix metalloproteinases (MMPs), especially MMP‐9, by oxidized low‐density lipoprotein (oxLDL) is implicated in many inflammatory diseases including brain injury. However, the signaling mechanisms underlying oxLDL‐induced MMP‐9 expression in astrocytes largely remain unknown. Here we report that oxLDL induces expression of proMMP‐9 via a MAPK‐dependent AP‐1 activation in rat brain astrocyte (RBA)‐1 cells. Results revealed by gelatin zymography, RT‐PCR, and Western blotting analyses showed that oxLDL‐induced proMMP‐9 gene expression was mediated through Akt, JNK1/2, and p42/p44 MAPK phosphorylation in RBA‐1 cells. These responses were attenuated by inhibitors of PI3K (LY294002), JNK (SP600125), and p42/p44 MAPK (PD98059), or transfection with dominant negative mutants and short hairpin RNA. Moreover, we demonstrated that AP‐1 (i.e., c‐Fos/c‐Jun) is crucial for oxLDL‐induced proMMP‐9 expression which was attenuated by pretreatment with AP‐1 inhibitor (curcumin). The regulation of MMP‐9 gene transcription by AP‐1 was confirmed by oxLDL‐stimulated MMP‐9 luciferase activity which was totally lost in cells transfected with the AP‐1 binding site‐mutated MMP‐9 promoter construct (mt‐AP1‐MMP‐9). These results suggested that oxLDL‐induced proMMP‐9 expression is mediated through PI3K/Akt, JNK1/2, and p42/p44 MAPK leading to AP‐1 activation. Understanding the regulatory mechanisms underlying oxLDL‐induced MMP‐9 expression in astrocytes might provide a new therapeutic strategy of brain injuries and diseases.

IL‐1β induces proMMP‐9 expression via c‐Src‐dependent PDGFR/PI3K/Akt/p300 cascade in rat brain astrocytes

In a previous study, interleukin‐1β (IL‐1β) has been shown to induce matrix metalloproteinases (MMPs) expression through mitogen‐activated protein kinases and nuclear factor‐κB pathways in rat brain astrocytes. Moreover, transactivation of growth factor receptors and phosphatidylinositol 3‐kinase (PI3K)/Akt cascade has been mentioned in the expression of several inflammatory genes. Here, we first report that IL‐1β‐induced up‐regulation of proMMP‐9 was inhibited by genistein. IL‐1β also stimulated phosphorylation of several protein tyrosine kinases such as c‐Src and platelet‐derived growth factor receptor (PDGFR), which was further confirmed by western blotting using an anti‐phospho‐c‐Src or anti‐phospho‐PDGFR antibody, respectively. IL‐1β‐stimulated c‐Src, PDGFR, and Akt phosphorylation and proMMP‐9 expression were attenuated by the inhibitors of c‐Src (PP1), PDGFR (AG1296), and PI3K (LY294002), respectively, or transfection with dominant negative plasmid of c‐Src or short hairpin RNAs of PDGFR and Akt. Moreover, IL‐1β‐induced proMMP‐9 expression was blocked by pre‐treatment with curcumin (a p300 inhibitor). We further confirmed that IL‐1β stimulated p300 recruitment to MMP‐9 promoter, and then acetylated histone H4 by immunoprecipitation and chromatin immunoprecipitation–PCR assays. The recruitment and activation of p300 in MMP‐9 promoter were inhibited by pre‐treatment with PP1, AG1296, and LY294002, respectively. Moreover, IL‐1β stimulated the c‐Src‐dependent transactivation of PDGFR/PI3K/Akt cascade is independent of nuclear factor‐κB pathway. These results indicated that in rat brain astrocytes cells, PI3K/Akt activation was mediated through c‐Src‐dependent transactivation of PDGFR promoted transcriptional co‐factor p300 recruitment and activation and eventually led to increased proMMP‐9 expression by IL‐1β.

Sphingosine 1‐phosphate induces EGFR expression via Akt/NF‐κB and ERK/AP‐1 pathways in rat vascular smooth muscle cells

Sphingosine 1‐phosphate (S1P) has been shown to regulate expression of several genes in vascular smooth muscle cells (VSMCs) and contributes to arteriosclerosis. However, the mechanisms regulating epidermal growth factor receptor (EGFR) expression by S1P in aortic VSMCs remain unclear. Western blotting and RT‐PCR analyses showed that S1P induced EGFR mRNA and protein expression in a time‐ and concentration‐dependent manner, which was attenuated by inhibitors of MEK1/2 (U0126) and phosphatidylinositide 3‐kinase (PI3K; wortmannin), and transfection with dominant negative mutants of ERK and Akt, respectively. These results suggested that S1P‐induced EGFR expression was mediated through p42/p44 MAPK and PI3K/Akt pathways in VSMCs. In accordance with these findings, S1P stimulated phosphorylation of p42/p44 MAPK and Akt which was attenuated by U0126 and wortmannin, respectively. Furthermore, S1P‐induced EGFR upregulation was blocked by a selective NF‐κB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P‐induced activation of NF‐κB was blocked by wortmannin, but not by U0126, suggesting that activation of NF‐κB was mediated through PI3K/Akt. Moreover, S1P‐induced EGFR expression was inhibited by an AP‐1 inhibitor curcumin and tanshinone IIA. S1P‐stimulated AP‐1 subunits (c‐Jun and c‐Fos mRNA) expression was attenuated by U0126 and wortmannin, suggesting that MEK and PI3K/ERK cascade linking to AP‐1 was involved in EGFR expression. Upregulation of EGFR by S1P may exert a phenotype modulation of VSMCs. This hypothesis was supported by pretreatment with AG1478 or transfection with shRNA of EGFR that attenuated EGF‐stimulated proliferation of VSMCs pretreated with S1P, determined by XTT assay. These results demonstrated that in VSMCs, activation of Akt/NF‐κB and ERK/AP‐1 pathways independently regulated S1P‐induced EGFR expression in VSMCs. Understanding the mechanisms involved in S1P‐induced EGFR expression on VSMCs may provide potential therapeutic targets in the treatment of arteriosclerosis. J. Cell. Biochem. 103: 1732–1746, 2008.

Functional coupling expression of COX-2 and cPLA2 induced by ATP in rat vascular smooth muscle cells: role of ERK1/2, p38 MAPK, and NF-κB

AIMS Vascular smooth muscle cells (VSMCs) that function as synthetic units play important roles in inflammatory diseases such as atherosclerosis and angiogenesis. As extracellular nucleotides such as ATP have been shown to act via activation of P(2) purinoceptors implicated in various inflammatory diseases, we hypothesized that extracellular nucleotides contribute to vascular diseases via upregulated expression of inflammatory proteins, such as cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) in VSMCs. METHODS AND RESULTS Western blotting, promoter assay, RT-PCR, and PGE2 immunoassay revealed that ATPgammaS induced expression of COX-2 and prostaglandin (PGE2) synthesis through the activation of p42/p44 MAPK (mitogen-activated protein kinase), p38 MAPK, and nuclear factor-kappaB (NF-kappaB). These responses were attenuated by inhibitors of MAPK/ERK kinase (MEK1/2; U0126), p38 MAPK (SB202190), and NF-kappaB (helenalin), or by tranfection with dominant negative mutants of p42, p38, IkappaB kinase (IKK)alpha, and IKKbeta. Furthermore, the ATPgammaS-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaBalpha was blocked by U0126 and helenalin. In addition, the ATPgammaS-stimulated cPLA2 expression was inhibited by U0126, SB202190, helenalin, celecoxib (a selective COX-2 inhibitor), and PGE2 receptor antagonists (AH6809, GW627368X, and SC-19220). However, the inhibitory effect of celecoxib on cPLA2 expression was reversed by addition of exogenous PGE2. CONCLUSION Our results suggest that in VSMCs, activation of p42/p44 MAPK, p38 MAPK, and NF-kappaB is essential for ATPgammaS-induced COX-2 expression and PGE2 synthesis. Newly synthesized PGE2 was observed to act as an autocrine signal contributing to cPLA2 expression, which may be implicated in inflammatory responses. Collectively, our findings provide insights into the correlation between COX-2 and cPLA2 expression in ATPgammaS-stimulated VSMCs with similar molecular mechanisms and functional coupling to amplify the occurrence of vessel disease-related vascular inflammation.