Suppression of Heme Oxygenase-1 by Prostaglandin E2-Protein Kinase A-A-Kinase Anchoring Protein Signaling Is Central for Augmented Cyclooxygenase-2 Expression in Lipopolysaccharide-Stimulated RAW 264.7 Macrophages.

PURPOSE: Prostaglandin (PG) E2 is an immunomodulatory lipid mediator generated mainly via the cyclooxygenase-2 (COX-2) pathway from arachidonic acid at sites of infection and inflammation. A positive feedback loop of PGE2 on COX-2 expression is critical for homeostasis during toll-like receptor (TLR)-mediated inflammatory processes. The mechanism of PGE2-regulated COX-2 expression remains poorly understood. The low-molecular-weight stress protein heme oxygenase-1 (HO-1) contributes to the anti-inflammatory, anti-oxidant and anti-apoptotic response against environmental stress. METHODS: We explored the involvement of HO-1 on PGE2 regulation of LPS-induced COX-2 expression in RAW 264.7 macrophages. RESULTS: LPS-induced COX-2 expression in RAW 264.7 macrophages was enhanced by exogenous PGE2 or cyclic AMP (cAMP) analogue and was suppressed by a COX inhibitor (indomethacin), a protein kinase A (PKA) inhibitor (KT5720), and A kinase anchoring protein (AKAP) disruptors (Ht31 and RIAD). This result suggests that the stimulatory effects of endogenous and exogenous PGE2 on COX-2 expression are mediated by a cAMP-PKA-AKAP-dependent pathway. The induction of HO-1 was observed in LPS-stimulated RAW 264.7 macrophages. This induction was suppressed by exogenous PGE2 and enhanced by blockage of the endogenous PGE2 effect by the PKA inhibitor or AKAP disruptors. In addition, HO-1 induction by the HO activator copper protoporphyrin suppressed LPS-induced COX-2 expression, which was restored by the addition of exogenous PGE2. The induction of HO-1 inhibited LPS-induced NF-κB p-65 nuclear expression and translocation. CONCLUSIONS: AKAP plays an important role in PGE2 regulation of COX-2 expression, and the suppression of HO-1 by PGE2-cAMP-PKA-AKAP signaling helps potentiate the LPS-induced COX-2 expression through a positive feedback loop in RAW 264.7 macrophages.

Comparison of explant-derived and enzymatic digestion-derived MSCs and the growth factors from Wharton's jelly.

Wharton's jelly is not only one of the most promising tissue sources for mesenchymal stem cells (MSCs) but also a source of natural growth factors. To prove that we can get both natural growth factors and MSCs from Wharton's jelly, we compared cellular characteristics and the level of basic fibroblast growth factor (bFGF) from samples using the explant culture method to those derived from the traditional enzymatic culture method. The levels of bFGF were 27.0 ± 11.7 ng/g on day 3, 15.6 ± 11.1 ng/g on day 6, and decreased to 2.6 ± 1.2 ng/g on day 14. The total amount of bFGF released was 55.0 ± 25.6 ng/g on explant culture. Compared with the traditional enzymatic digestion method, the explant culture method showed a tendency to release higher levels of bFGF in supernatant media for the first week of culture, and the higher cellular yield at passage 0 (4.89 ± 3.2 × 10(5)/g versus 1.75 ± 2.2 × 10(5)/g, P = 0.01). In addition, the genes related to mitosis were upregulated in the explant-derived MSCs.

Introducing pulsed low-intensity ultrasound to culturing human umbilical cord-derived mesenchymal stem cells.

The human umbilical cord (hUC) is a source of adult tissue-derived mesenchymal stem cells (MSCs). A pulsed low-intensity ultrasound (PLIUS) method is described for increasing the yield of MSCs from whole hUC without enzymatic digestion or growth factor supplementation. Analysis of the immunophenotype of cells and a differentiation study were performed to show the compatibility of MSCs. The mean number of cells recovered from primocultures of hUC was 6 x 10(5) cells/cm. PLIUS resulted in a 3.3-fold increase in MSC yield at passage 0. PLIUS exposure increases the yield of hUC-MSCs by promoting release and enhancing proliferation.

Chondrogenic differentiation of mesenchymal stem cells isolated from patients in late adulthood: the optimal conditions of growth factors.

There is a controversy about the capacity of the mesenchymal stem cells (MSCs) from aged individuals to proliferate and differentiate into cartilage. The purpose of this study was to investigate the optimal condition to culture human MSCs from the aged individuals (>50 years) for cartilage tissue engineering. We tested the hypothesis that effective proliferation and chondrogenesis can be achieved with human MSCs from aged individuals under appropriate conditions. To investigate the best condition for proliferation, MSCs were cultured in medium containing four concentrations subsets (0, 0.05, 0.5, 5 ng/mL) of recombinant human TGF-beta2 and FGF-2, either with or without fetal calf serum. The cell numbers were counted 0, 1, 3, and 7 days after growth factors were given. For the induction of chondrogenesis in 3-dimensional (3-D) culture, cells were cultured in pellets with chondrogenic medium containing combinations of various growth factors. After 4 weeks of culture, the pellets were fixed and evaluated with Safranin-O staining for proteoglycan and immunohistochemical staining for type II collagen. RT-PCR was also performed for the mRNAs of type I collagen, type II collagen, and cartilage oligomeric protein (COMP). In a monolayer culture, TGF-beta2 in concentrations of 0.5 and 5 ng/mL caused significant reduction in cell number irrespective of the presence of serum. FGF-2 of 5 ng/mL most effectively increased cell number even in the absence of serum. In a pellet culture, remarkable chondrocyte-like differentiation of cells was induced around the peripheral areas of a pellet with 5 ng/mL of TGF-beta2, accompanied by increased proteoglycan and type II collagen production. The addition of 100 ng/mL of IGF-I induced notable increase in proteoglycan contents. The results of RT-PCR mirrored those of histological studies. This study shows that an effective proliferation and chondrogenesis may be obtained with proper combinations of growth factors and mesenchymal stem cells from aged individuals.

Heme oxygenase-1-mediated partial cytoprotective effect by NO on cadmium-induced cytotoxicity in C6 rat glioma cells.

Heme oxygenase-1 (HO-1) is a 32-kDa stress induced enzyme that degrades heme to carbon monoxide (CO) and biliverdin. By employing RT-PCR and Western blotting techniques, we have examined the HO-1 induction in C6 glioma cells that were treated with cadmium chloride (CdCl(2)) or spermine NONOate (SPER/NO). By employing a cell viability assay, we have also examined the cytoprotective effect of HO-1 induction against the cytotoxicity caused by toxic dose of CdCl(2). In C6 glioma cells exposed to CdCl(2), expression of HO-1 (mRNA and protein) was increased in a dose- and time-dependent manner. Nitric oxide (NO) generated from SPER/NO very rapidly increased HO-1 mRNA expression in the C6 glioma cells. The induction of HO-1 by SPER/NO protected the cells from toxic dose of CdCl(2). The up-regulation of HO-1 mRNA expression by CdCl(2) was inhibited by a pre-incubation of the cells with actinomycin D, a potent inhibitor of mRNA transcription. Upon the inhibition of elevated HO-1 mRNA expression by the use of zinc protoporphyrin IX (ZnPP), an inhibitor of HO activity, the change of HO-1 mRNA expression by ZnPP was not observed. Thus, the glial cell may respond to CdCl(2) toxicity by enhancing the HO-1 expression in its effort to minimize the CdCl(2)-derived oxidative damage, and to survive. In the glioma cells, when the HO-1 expression was elevated by a prior incubation with SPER/NO, the cell viability against the cytotoxicity of CdCl(2) was significantly increased. When the results of our experiment are taken together, we discovered that NO provided a rapid enhancement of HO-1 expression, and it provided a protective effect against CdCl(2)-derived oxidative injury in the C6 rat glioma cells.

Evidence for cyclooxygenase-1 association with caveolin-1 and -2 in cultured human embryonic kidney (HEK 293) cells.

The purpose of this study was to confirm protein-protein interaction between cyclooxygenase-1 (COX-1) and caveolins. The interaction of cyclooxygenase-1 and caveolins in the cultured human embryonic kidney (HEK 293) cells was investigated using immuno-precipitation and Western blot analysis. In HEK 293 cells, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Caveolae rich membranous fractions from the HEK 293 cells contained both COX-1 and caveolin-1 or caveolin-2 in same fractions. The experiments of immuno-precipitation showed complex formation between the COX-1 and caveolin-1 or caveolin-2 in the HEK 293 cells. Confocal microscopic results also support co-localization of COX-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with cylooxygenase-1 in caveolae suggested that caveolin would play an important role in regulating the function of COX-1.

Evidence for heme oxygenase-1 association with caveolin-1 and -2 in mouse mesangial cells.

The interaction of heme oxygenase-1 (HO-1) and caveolin in the cultured mouse mesangial cells (MMC) was investigated. In normal MMCs, high levels of caveolin-2 and low level of caveolin-1 at mRNA and protein level were observed without any detectable expression of caveolin-3. Upon treating the MMCs either with cadmium (Cd) or spermine NONOate (SPER/NO), expression of HO-1 mRNA and protein was increased. Caveolae rich membranous fractions from the MMCs treated with Cd or SPER/NO contained both HO-1 and caveolin-1 or caveolin-2. The experiments of immuno-precipitation showed complex formation between the HO-1 and caveolin-1 or caveolin-2 in the Cd treated MMCs. Confocal microscopic results also support co-localization of HO-1 and caveolin-1 or caveolin-2 at the plasma membrane. Co-localization of caveolins with HO-1 in caveolae suggested that caveolin could also play an important role in regulating the function of HO-1.

Down-regulation of organic anion transporter 2 mRNA expression by nitric oxide in primary cultured rat hepatocytes.

Role of nitric oxide (NO) on the expression of organic anion transporter 2 (OAT2) located in sinusoidal domain of hepatocytes has been investigated. Effect of NO generated in vivo in rat and delivered in vitro to hepatocytes was determined. Lipopolysaccharide (LPS) and spermine NONOate (SPER/NO) were selected as the NO donors for in vivo and in vitro experiments, respectively. Constitutive basal expression of rOAT2 mRNA was detected in the normal rat liver and the level of its expression was decreased by intraperitoneal administration of LPS. This LPS-induced decrement did not occur when aminoguanidine (AG), an inhibitor of iNOS, was co-administered with LPS. The expression of rOAT2 mRNA was detected in hepatocytes, but not in the nonparenchymal cells. In the primary cultured hepatocytes obtained from normal rats (normal hepatocytes), a time-dependent decline of rOAT2 mRNA expression was observed, but not in the hepatocytes obtained from rats pretreated with gadolinium chloride (GdCl3, Gd-hepatocytes), an inhibitor of Kupffer cell activation. The decline of rOAT2 mRNA expression observed in the normal hepatocytes was enhanced by LPS treatment, but not in the Gd-hepatocytes. The LPS-dependent enhancement in the decline of rOAT2 mRNA expression did not occur when the normal hepatocytes were treated with AG or actinomycin D. When the Gd-hepatocytes were treated with SPER/NO, an NO donor, the rOAT2 mRNA expression declined markedly. Combined, our results suggest that rOAT2 mRNA expression in hepatocytes is down-regulated by NO at least at the transcriptional step.