Protein kinase C-delta transactivates platelet-derived growth factor receptor-alpha in mechanical strain-induced collagenase 3 (matrix metalloproteinase-13) expression by osteoblast-like cells.

Matrix metalloproteinase-13 (MMP-13, or collagenase 3) has been shown to degrade intact collagen and to participate in situations where rapid and effective remodeling of collagenous ECM is required. Mechanical strain induction of MMP-13 is an example of how osteoblasts respond to high mechanical forces and participate in the bone-remodeling mechanism. Using MC3T3-E1 osteoblast-like cells, we dissected the signaling molecules involved in MMP-13 induction by mechanical strain. Reverse transcription-PCR and zymogram analysis showed that platelet-derived growth factor receptor (PDGFR) inhibitor, AG1296, inhibited the mechanical strain-induced MMP-13 gene and activity. However, the induction was not affected by anti-PDGF-AA serum. Immunoblot analysis revealed time-dependent phosphorylation of PDGFR-alpha up to 2.7-fold increases within 3 min under strain. Transfection with shPDGFR-alpha (at 4 and 8 microg/ml) abolished PDGFR-alpha and reduced MMP-13 expression. Moreover, time-dependent recruitments of phosphoinositide 3-kinase (PI3K) by PDGFR-alpha were detected by immunoprecipitation with anti-PDGFR-alpha serum followed by immunoblot with anti-PI3K serum. AG1296 inhibited PDGFR-alpha/PI3K aggregation and Akt phosphorylation. Interestingly, protein kinase C-delta (PKC-delta) inhibitor, rottlerin, inhibited not only PDGFR-alpha/PI3K aggregation but PDGFR-alpha phosphorylation. The sequential activations were further confirmed by mutants DeltaPKC-delta, DeltaAkt, and DeltaERK1. Consistently, the primary mouse osteoblast cells used the same identified signaling molecules to express MMP-13 under mechanical strain. These results demonstrate that, in osteoblast-like cells, the MMP-13 induction by mechanical strain requires the transactivation of PDGFR-alpha by PKC-delta and the cross-talk between PDGFR-alpha/PI3K/Akt and MEK/ERK pathways.

Vertical skeletal and facial profile changes after surgical correction of mandibular prognathism.

BACKGROUND: Mandibular prognathism is often corrected by surgical orthodontics. Correction of the sagittal facial profile has received wide attention. However, vertical changes remained undefined and thus, were investigated. METHODS: Subjects included 18 patients with mandibular prognathism who had surgical correction (S group, mean age: 20.1 +/- 3.2 years) and 18 patients with Class I malocclusion (C group, mean age: 21.2 +/- 3.6 years). Cephalograms were taken at the initial visit (T1) for both the groups and one year after surgery (T2) for the S group and analyzed by standard protocols. The vertical differences between the S and C groups at T1 and within the S group at T1 and T2 were compared. Additionally, the C group at T1 and the S group at T2 were compared. RESULTS: Comparison between groups at T1 revealed no difference in the anterior and posterior upper facial heights (58 mm and 50 mm, respectively). However, the S group exhibited a longer anterior lower facial height and a shorter posterior lower facial height. Accordingly, any vertical measurements and comparisons related to the mandible revealed significant difference between groups. Surgical correction did not change the vertical chin position. Contrarily, the posterior ramus heights were reduced (from 54 to 50 mm). The vertical measurements and comparisons for soft tissues reflected those for hard tissues. CONCLUSIONS: The results indicate that through surgical correction of mandibular prognathism, vertical facial heights can be maintained within normal physiological function.

Lipopolysaccharide induces VCAM-1 expression and neutrophil adhesion to human tracheal smooth muscle cells: involvement of Src/EGFR/PI3-K/Akt pathway.

In our previous study, LPS has been shown to induce vascular cell adhesion molecule-1(VCAM-1) expression through MAPKs and NF-kappaB in human tracheal smooth muscle cells (HTSMCs). In addition to these pathways, the non-receptor tyrosine kinases (Src), EGF receptor (EGFR), and phosphatidylinositol 3-kinase (PI3K) have been shown to be implicated in the expression of several inflammatory target proteins. Here, we reported that LPS-induced up-regulation of VCAM-1 enhanced the adhesion of neutrophils onto HTSMC monolayer, which was inhibited by LY294002 and wortmannin. LPS stimulated phosphorylation of protein tyrosine kinases including Src, PYK2, and EGFR, which were further confirmed using specific anti-phospho-Src, PYK2, or EGFR Ab, respectively, revealed by Western blotting. LPS-stimulated Src, PYK2, EGFR, and Akt phosphorylation and VCAM-1 expression were attenuated by the inhibitors of Src (PP1), EGFR (AG1478), PI3-K (LY294002 and wortmannin), and Akt (SH-5), respectively, or transfection with siRNAs of Src or Akt and shRNA of p110. LPS-induced VCAM-1 expression was also blocked by pretreatment with curcumin (a p300 inhibitor) or transfection with p300 siRNA. LPS-stimulated Akt activation translocated into nucleus and associated with p300 and VCAM-1 promoter region was further confirmed by immunofluorescence, immunoprecipitation, and chromatin immunoprecipitation assays. This association of Akt and p300 to VCAM-1 promoter was inhibited by pretreatment with PP1, AG1478, wortmannin, and SH-5. LPS-induced p300 activation enhanced VCAM-1 promoter activity and VCAM-1 mRNA expression. These results suggested that in HTSMCs, Akt phosphorylation mediated through transactivation of Src/PYK2/EGFR promoted the transcriptional p300 activity and eventually led to VCAM-1 expression induced by LPS.

Sphingosine 1-phosphate induces EGFR expression via Akt/NF-kappaB 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-kappaB inhibitor helenalin. Immunofluorescent staining and reporter gene assay revealed that S1P-induced activation of NF-kappaB was blocked by wortmannin, but not by U0126, suggesting that activation of NF-kappaB 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-kappaB 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.

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-kappaB inhibitor helenalin. Consistently, S1P-stimulated translocation of NF-kappaB into the nucleus was revealed by immnofluorescence staining. Moreover, S1P-stimulated activation of NF-kappaB 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-kappaB. COX-2 promoter assay showed that S1P induced COX-2 promoter activity mediated through p42/p44 MAPK, PI3K/Akt, and NF-kappaB. These results suggested that in VSMCs, activation of p42/p44 MAPK, Akt and NF-kappaB 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.

Intrusion of the overerupted upper left first and second molars by mini-implants with partial-fixed orthodontic appliances: a case report.

Overeruption of maxillary molar(s) because of loss of the opposing teeth creates occlusal interference and functional disturbances. To restore proper occlusion, intrusion of the overerupted molars becomes essential before reconstruction can be initiated. A plausible procedure is orthodontic intrusion, which demands calibrated anchorage support from intraoral multiunit teeth and from headgear wear. In this report, we present a simplified and localized version of the orthodontic appliances in conjunction with mini-implants to intrude the overerupted molars. The purpose of using implants as skeletal anchorage was to eliminate the need for patient compliance for headgear wear and to overcome the difficulty resulting from the shortage of anchor teeth. The results showed that the biological responses of the teeth and the surrounding bony structures to the intrusion appeared normal and acceptable. Furthermore, the periodontal health and vitality of the teeth were well maintained even after a one-year follow-up.

Mechanical strain induces collagenase-3 (MMP-13) expression in MC3T3-E1 osteoblastic cells.

Mechanical strain plays a crucial role in bone remodeling during growth and development and healing of bone besides systemic and local factors. One of the major factors involves in remodeling process is matrix metalloproteinases (MMPs) such as MMP-13 that has been shown to degrade the native interstitial collagens in several tissues. To study how mechanical strain affects extracellular matrix degradation by MMP-13, a biaxial strain was applied to MC3T3-E1 osteoblastic cells plated onto a collagen-coated flexible elastic membrane. The MMP-13 protein and mRNA expression were determined by Western blotting and reverse transcriptase-PCR, respectively. The zymographic activities of MMP-13 increased dramatically at 30 min, reached a peak by 2-fold at 1 h, and maintained up to 4 h. Moreover, the MMP-13 and c-fos mRNA expressed at 5 min, increased to 2.8- and 3-fold at 1 h, respectively, and gradually declined thereafter. Cycloheximide and actinomycin D did not inhibit the MMP-13 and c-fos mRNA expression, suggesting that such expression does not require de novo protein synthesis and not change their stabilities. To investigate which of the mitogen-activated protein kinase (MAPK) pathways involves in the expression of MMP-13, inhibitors such as PD98059, SB203580, and SP600125 were used. However, only PD98059 (an inhibitor of MEK1/2 activation) inhibited MMP-13 and c-fos gene expression; the result was further substantiated by transfecting with the dominant negative mutants of MEK1/2 (MEK K97R) and ERK2. Taken together, our results showed that mechanical strain induces the MMP-13 expression through MEK-ERK signaling pathway to regulate mechanical adaptation.

Bradykinin B2 receptor-mediated proliferation via activation of the Ras/Raf/MEK/MAPK pathway in rat vascular smooth muscle cells.

It has been suggested that bradykinin (BK) plays an important role in regulating neointimal formation after vascular injury. However, implication of BK in the growth of rat vascular smooth muscle cells (VSMCs) is controversial. Therefore, we examined the mitogenic effect of BK on VSMCs associated with activation of mitogen-activated protein kinase (MAPK). Both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were activated by BK in time- and concentration-dependent manners. Pretreatment of these cells with neither pertussis toxin nor cholera toxin attenuated the BK-induced responses. Pretreatment of VSMCs with Hoe 140 (a selective B(2) receptor antagonist), U73122 (an inhibitor of phospholipase C), and BAPTA/AM (an intracellular Ca(2+) chelator) inhibited both [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation in response to BK. BK-induced [(3)H]thymidine incorporation and p42/p44 MAPK phosphorylation were inhibited by pretreatment of VSMCs with tyrosine kinase inhibitors (genistein and herbimycin A), protein kinase C (PKC) inhibitors (staurosporine, Go-6976, and Ro-318220), an MAPK kinase inhibitor (PD98059), and a p38 MAPK inhibitor (SB203580). Overexpression of the dominant negative mutants, H-Ras-15A and Raf-N4, suppressed p42/p44 MAPK activation induced by BK and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. From these results, we concluded that the mitogenic effect of BK is mediated through activation of the Ras/Raf/MEK/MAPK pathway similar to that of PDGF-BB. BK-mediated MAPK activation was modulated by Ca(2+), PKC, and tyrosine kinase all of which are associated with cell proliferation in rat cultured VSMCs.