Rong-Sen Yang

Adiponectin Enhances IL-6 Production in Human Synovial Fibroblast via an AdipoR1 Receptor, AMPK, p38, and NF-κB Pathway

Articular adipose tissue is a ubiquitous component of human joints, and adiponectin is a protein hormone secreted predominantly by differentiated adipocytes and involved in energy homeostasis. We investigated the signaling pathway involved in IL-6 production caused by adiponectin in both rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts. Rheumatoid arthritis synovial fibroblasts and osteoarthritis synovial fibroblasts expressed the AdipoR1 and AdipoR2 isoforms of the adiponectin receptor. Adiponectin caused concentration- and time-dependent increases in IL-6 production. Adiponectin-mediated IL-6 production was attenuated by AdipoR1 and 5′-AMP-activated protein kinase (AMPK)α1 small interference RNA. Pretreatment with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-κB inhibitor, IκB protease inhibitor, and NF-κB inhibitor peptide also inhibited the potentiating action of adiponectin. Adiponectin increased the kinase activity and phosphorylation of AMPK and p38. Stimulation of synovial fibroblasts with adiponectin activated IκB kinase α/β (IKK α/β), IκBα phosphorylation, IκBα degradation, p65 phosphorylation at Ser (276), p65 and p50 translocation from the cytosol to the nucleus, and κB-luciferase activity. Adiponectin-mediated an increase of IKK α/β activity, κB-luciferase activity, and p65 and p50 binding to the NF-κB element and was inhibited by compound C, SB203580 and AdipoR1 small interference RNA. Our results suggest that adiponectin increased IL-6 production in synovial fibroblasts via the AdipoR1 receptor/AMPK/p38/IKKαβ and NF-κB signaling pathway.

High Glucose Induces Human Endothelial Cell Apoptosis Through a Phosphoinositide 3-Kinase–Regulated Cyclooxygenase-2 Pathway

Objectives—Diabetes mellitus causes endothelial dysfunction. The precise molecular mechanisms by which hyperglycemia causes apoptosis in endothelial cells are not yet well understood. The aim of this study was to explore the role of cyclooxygenase-2 (COX-2) and the possible involvement of phosphoinositide 3-kinase (PI3K) signaling in high glucose (HG)–induced apoptosis in human umbilical vein endothelial cells (HUVECs). Methods and Results—For detection of apoptosis, the morphological Hoechst staining and Annexin V/propidium iodide staining were used. Glucose upregulated COX-2 protein expression, which was associated with the induction of prostaglandin (PG) E2 (PGE2), caspase-3 activity, and apoptosis. Unexpectedly, we found that PI3K inhibitors could suppress COX-2 expression, PGE2 production, caspase-3 activity, and the subsequent apoptosis under HG condition. Glucose-induced activation of PI3K resulted in the downstream effector Akt phosphorylation. PI3K inhibitors effectively attenuated the intracellular reactive oxygen species (ROS) generation and nuclear factor &kgr;B (NF-&kgr;B) activation. Blocking the PI3K and Akt activities with the dominant-negative vectors greatly diminished the HG-triggered NF-&kgr;B activation and COX-2 expression and apoptosis. Conclusions—These results suggest that HG, via PI3K/Akt signaling, induces NF-&kgr;B–related upregulation of COX-2, which in turn triggers the caspase-3 activity that facilitates HUVEC apoptosis. Also, HG may cause ROS generation in HUVECs through a PI3K/Akt–dependent pathway.

Ultrasound stimulates cyclooxygenase-2 expression and increases bone formation through integrin, focal adhesion kinase, phosphatidylinositol 3-kinase, and Akt pathway in osteoblasts.

It has been shown that ultrasound (US) stimulation accelerates fracture healing in animal models and in clinical studies. Here we found that US stimulation transiently increased the surface expression of α2, α5, β1, and β3 integrins in cultured osteoblasts, as shown by flow cytometric analysis and immunofluorescence staining. US stimulation increased prostaglandin E2 formation and the protein and mRNA levels of cyclooxygenase-2 (COX-2). At the mechanistic level, anti-integrin α5β1 and αvβ3 antibodies or rhodostomin, a snake venom disintegrin, attenuated the US-induced COX-2 expression. Phosphatidylinositol 3-kinase (PI3K) inhibitors 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride (LY294002) and wortmannin also inhibited the potentiating action of US. US stimulation increased the phosphorylation of focal adhesion kinase (FAK), extracellular signal-regulated kinases (ERK), p85 subunit of PI3K, and serine 473 of Akt. COX-2 promoter activity was enhanced by US stimulation in cells transfected with pCOX2-Luc. Cotransfection with dominant-negative mutant of FAK(Y397F), p85(Δp85), Akt(K179A), or ERK2(K52R) inhibited the potentiating action of US on COX-2 promoter activity. Expression of mineralized nodule was lower in dominant-negative mutants of FAK, p85, and Akt-transfected clones than in vector-transfected control cells. Taken together, our results provide evidence that US stimulation increases COX-2 expression and promotes bone formation in osteoblasts via the integrin/FAK/PI3K/Akt and ERK signaling pathway.

Expandable endoprosthesis reconstruction in skeletally immature patients with tumors.

Between September 1984 and January 1996, 32 expandable endoprostheses were used for limb reconstruction after resection of malignant bone tumors in patients who were skeletally immature. The 20 boys and 12 girls ranged in age from 3 to 15 years (mean, 9.7 years). One patient had a Stage IIA tumor, 22 patients had Stage IIB tumors, and seven patients had Stage III tumors according to the classification of the Musculoskeletal Tumor Society. There also were two patients with parosteal osteosarcomas. The histologic diagnosis was osteosarcoma in 23 patients and Ewings sarcoma in nine. All patients except the patients with parosteal osteosarcoma received standard neoadjuvant therapy. Twenty-two Lewis Expandable Adjustable Prostheses, four modular Wright Medical prostheses, four modular Howmedica prostheses, and two Techmedica expandable prostheses were used. Thirteen patients died, two have no evidence of disease, and 17 are continuously disease free. Sixteen of 32 patients (50%) have not had an expansion procedure because of early death in 10 and early amputation in three. Three patients are waiting to undergo an expansion procedure. Sixteen of the 32 patients (50%) have undergone 32 expansion procedures, to a maximum of 9 cm, without any infection. To maintain range of motion before the expansion procedure, a complete resection of the pseudocapsule was done routinely. Fourteen of the 32 patients did not have complications. Eighteen of the 32 patients had 27 complications. All Lewis Expandable Adjustable Prosthesis endoprostheses and the two nonmodular Techmedica prostheses were associated with a large amount of titanium debris. The childrens functional results were similar to the results reported for adults with an average Musculoskeletal Tumor Society rating of good to excellent at the knee, fair to good at the hip, and fair about the shoulder. Rehabilitation of the knee in very young patients (5-8 years) remains problematic and careful selection of patient and family is necessary. The Lewis Expandable Adjustable Prosthesis probably should be reserved for very young patients (5-8 years) and modular systems should be used for large preadolescent and adolescent children.

Leptin-Induced IL-6 Production Is Mediated by Leptin Receptor, Insulin Receptor Substrate-1, Phosphatidylinositol 3-Kinase, Akt, NF-κB, and p300 Pathway in Microglia

Leptin, the adipocyte-secreted hormone that centrally regulates weight control, is known to function as an immunomodulatory regulator. We investigated the signaling pathway involved in IL-6 production caused by leptin in microglia. Microglia expressed the long (OBRl) and short (OBRs) isoforms of the leptin receptor. Leptin caused concentration- and time-dependent increases in IL-6 production. Leptin-mediated IL-6 production was attenuated by OBRl receptor antisense oligonucleotide, PI3K inhibitor (Ly294002 and wortmannin), Akt inhibitor (1L-6-hydroxymethyl-chiro-inositol-2-((R)-2-O-methyl-3-O-octadecylcarbonate)), NF-κB inhibitor (pyrrolidine dithiocarbamate), IκB protease inhibitor (l-1-tosylamido-2-phenylenylethyl chloromethyl ketone), IκBα phosphorylation inhibitor (Bay 117082), or NF-κB inhibitor peptide. Transfection with insulin receptor substrate (IRS)-1 small-interference RNA or the dominant-negative mutant of p85 and Akt also inhibited the potentiating action of leptin. Stimulation of microglia with leptin activated IκB kinase α/IκB kinase β, IκBα phosphorylation, IκBα degradation, p65 phosphorylation at Ser276, p65 and p50 translocation from the cytosol to the nucleus, and κB-luciferase activity. Leptin-mediated an increase of IκB kinase α/IκB kinase β activity, κB-luciferase activity, and p65 and p50 binding to the NF-κB element was inhibited by wortmannin, Akt inhibitor, and IRS-1 small-interference RNA. The binding of p65 and p50 to the NF-κB elements, as well as the recruitment of p300 and the enhancement of histone H3 and H4 acetylation on the IL-6 promoter was enhanced by leptin. Our results suggest that leptin increased IL-6 production in microglia via the leptin receptor/IRS-1/PI3K/Akt/NF-κB and p300 signaling pathway.

Efficacy of a 24-Week Aerobic Exercise Program for Osteopenic Postmenopausal Women

Abstract. Osteoporosis is one of the most common skeletal disorders affecting postmenopausal women. The purpose of this study was to investigate whether a 24-week program of aerobic high-impact loading exercise was beneficial for enhancing physical fitness and bone mineral density (BMD) in osteopenic postmenopausal women. Forty-three postmenopausal women aged 48–65 years participated in this study. The BMD of the spine (L2–L4) and right femoral neck of each woman was below 1 SD of the mean of premenopausal women, as examined by dual X-ray absorptiometry. The assignment of subjects into exercise or control group was not randomized but based on each subjects anticipated compliance to the 6-month long exercise program. Twenty-two subjects joined the exercise group and attended the training programs and 21 served as the control group. Exercise programs included treadmill walking at an intensity above 70% of maximal oxygen consumption (VO2max) for 30 minutes, followed by 10 minutes of stepping exercise using a 20-cm-high bench. The program was conducted three times per week for 24 weeks. Physical fitness measurements included testing of flexibility, muscular strength and endurance, body composition, and cardiopulmonary fitness. The results showed that the quadriceps strength, muscular endurance, and VO2max in the exercise group had significant improvements, whereas no improvement was found in any of the physical fitness parameters in the control group. The BMD of the L2–L4 and the femoral neck in the exercise group increased 2.0% (P > 0.05) and 6.8% (P < 0.05) and those in the control group decreased 2.3% (P < 0.05) and 1.5% (P > 0.05), respectively. In conclusion, aerobics combined with high-impact exercise at a moderate intensity was effective in offsetting the decline in BMD in osteopenic postmenopausal women.

Prostaglandin E2 Stimulates Fibronectin Expression through EP1 Receptor, Phospholipase C, Protein Kinase Cα, and c-Src Pathway in Primary Cultured Rat Osteoblasts

Fibronectin (Fn) is involved in the early stages of bone formation, and prostaglandin E (PGE) is an important factor regulating osteogenesis. Here we found that PGE2 enhanced extracellular Fn assembly in rat primary osteoblasts, as shown by immunofluorescence staining and enzyme-linked immunosorbent assay. PGE2 also increased the protein levels of Fn by using Western blotting analysis. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptor, antisense oligonucleotides revealed that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated up-regulation of Fn. At the mechanistic level, Ca2+ chelator (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl ester)), phosphatidylinositol-phospholipase C inhibitor (U73122), or Src inhibitor (PP2) attenuated the PGE2-induced Fn expression. Protein kinase C (PKC) inhibitor (GF109203X) also inhibited the potentiating action of PGE2. Furthermore, treatment with antisense oligonucleotides of various PKC isoforms, including α, β, ϵ, and δ, demonstrated that α isozyme plays an important role in the enhancement action of PGE2 on Fn assembly. Flow cytometry and reverse transcription-PCR showed that PGE2 and 17-phenyl trinor PGE2 (EP1/EP3 agonist) increased the surface expression and mRNA level of α5 or β1 integrins. Fn promoter activity was enhanced by PGE2 and 17-phenyl trinor PGE2 in cells transfected with pGL2F1900-Luc. Cotransfection with dominant negative mutants of PKCα or c-Src inhibited the potentiating action of PGE2 on Fn promoter activity. Local administration of PGE2 or 17-phenyl trinor PGE2 into the metaphysis of the tibia via the implantation of a needle cannula significantly increased the Fn and α5β1 integrin immunostaining and bone volume of secondary spongiosa in tibia. Taken together, our results provided evidence that PGE2 increased Fn and promoted bone formation in rat osteoblasts via the EP1/phospholipase C/PKCα/c-Src signaling pathway.

Increased bone growth by local prostaglandin E2 in rats

SummaryThe effects of prostaglandin E2 (PGE2) on bone growth were investigated in rats. Daily injection of PGE2 (1, 10, and 100 pmol) was given via local intraosseous route into the metaphysis of the left tibia for 14 days. The contralateral right tibia injected with vehicle and saline was for the control. The rats receiving no injection provided as normal control. The results obtained indicated that PGE2 slightly but significantly decreased the body weight increment without effect on tibial length. The most prominent effect of PGE2 was the increase of metaphyseal bone trabeculae by 45–81% in a dose-dependent manner. The microscopic examination revealed that PGE2 unequivocally increased the new woven bone formation. The bone cell population study showed no difference between the number of osteoblasts and osteoclasts in primary spongiosa of the PGE2-injected limbs and those of contralateral limbs. However, the numbers of osteoblasts and osteoclasts were markedly increased in secondary spongiosa in the PGE2-injected limbs. This finding confirmed a stimulatory role of PGE2 in the bone formation. The local intraosseous injection of PGE2 was proven to be a good model for the study of local growth factors on bone metabolism with a lower effective dose which eliminates the systemic side effects.

The Role of Phosphoinositide 3-Kinase/Akt Signaling in Low-Dose Mercury–Induced Mouse Pancreatic β-Cell Dysfunction In Vitro and In Vivo

The relationship between oxidation stress and phosphoinositide 3-kinase (PI3K) signaling in pancreatic β-cell dysfunction remains unclear. Mercury is a well-known toxic metal that induces oxidative stress. Submicromolar-concentration HgCl2 or methylmercury triggered reactive oxygen species (ROS) production and decreased insulin secretion in β-cell–derived HIT-T15 cells and isolated mouse islets. Mercury increased PI3K activity and its downstream effector Akt phosphorylation. Antioxidant N-acetyl-l-cysteine (NAC) prevented mercury-induced insulin secretion inhibition and Akt phosphorylation but not increased PI3K activity. Inhibition of PI3K/Akt activity with PI3K inhibitor or by expressing the dominant-negative p85 or Akt prevented mercury-induced insulin secretion inhibition but not ROS production. These results indicate that both PI3K and ROS independently regulated Akt signaling–related, mercury-induced insulin secretion inhibition. We next observed that 2- or 4-week oral exposure to low-dose mercury to mice significantly caused the decrease in plasma insulin and displayed the elevation of blood glucose and plasma lipid peroxidation and glucose intolerance. Akt phosphorylation was shown in islets isolated from mercury-exposed mice. NAC effectively antagonized mercury-induced responses. Mercury-induced in vivo effects and increased blood mercury were reversed after mercury exposure was terminated. These results demonstrate that low-dose mercury–induced oxidative stress and PI3K activation cause Akt signaling–related pancreatic β-cell dysfunction.

Stromal Cell-Derived Factor-1 Induces Matrix Metalloprotease- 13 Expression in Human Chondrocytes

The production of chemokine stromal cell-derived factor (SDF)-1 is significantly higher in synovial fluid of patients with osteoarthritis and rheumatoid arthritis. Matrix metalloproteinase (MMP)-13 may contribute to the breakdown of articular cartilage during arthritis. Here, we found that SDF-1α increased the secretion of MMP-13 in cultured human chondrocytes, as shown by reverse transcriptase-polymerase chain reaction, Western blot, and zymographic analysis. SDF-1α also increased the surface expression of CXCR4 receptor in human chondrocytes. CXCR4-neutralizing antibody, CXCR4-specific inhibitor [1-[[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methyl]-1,4,8,11-tetrazacyclotetradecane (AMD3100)], or small interfering RNA against CXCR4 inhibited the SDF-1α-induced increase of MMP-13 expression. The transcriptional regulation of MMP-13 by SDF-1α was mediated by phosphorylation of extracellular signal-regulated kinases (ERK) and activation of the activator protein (AP)-1 components of c-Fos and c-Jun. The binding of c-Fos and c-Jun to the activator protein (AP-1) element on the MMP-13 promoter and the increase in luciferase activity was enhanced by SDF-1α. Cotransfection with dominant-negative mutant of ERK2 or c-Fos and c-Jun antisense oligonucleotide inhibited the potentiating action of SDF-1α on MMP-13 promoter activity. Taken together, our results provide evidence that SDF-1α acts through CXCR4 to activate ERK and the downstream transcription factors (c-Fos and c-Jun), resulting in the activation of AP-1 on the MMP-13 promoter and contributing cartilage destruction during arthritis.