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Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Fox B, Schantz JT, Haigh R, Wood ME, Moore PK, Viner N, Spencer JP, Winyard PG, Whiteman M - J. Cell. Mol. Med. (2012)

Bottom Line: Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment.These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs.H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Peninsula Medical School, University of Exeter, St. Luke's Campus, Exeter, Devon, UK.

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Inducible expression and activity of CSE but not CBS in human chondrogenically differentiated mesenchymal progenitor cells (MPCs). (A–C) CSE and CBS protein expression determined by Western blotting and (D, E) Western blot analysis by densitometry. (F) Cytokine induced H2S synthesis in MPC. Cells were treated with TNF-α, IL-1β or IL-6 at a final concentration of 5 ng/ml for 18 hrs. Cells were lysed with RIPA buffer and 20 μg protein analysed by Western blotting for CSE, CBS and β-actin expression. H2S synthesis was determined by zinc-trap specrophotometry in the presence and absence of D,L-propargylglycine (PAG; to inhibit CSE) or aminooxyacetate (AOAA; to inhibit CBS) added a final concentration of 1 mmol/l as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. Figure 1(D) ***P < 0.001, cf. untreated cells; Figure 1(F) *P < 0.05, **P < 0.01, ***P < 0.001.
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fig01: Inducible expression and activity of CSE but not CBS in human chondrogenically differentiated mesenchymal progenitor cells (MPCs). (A–C) CSE and CBS protein expression determined by Western blotting and (D, E) Western blot analysis by densitometry. (F) Cytokine induced H2S synthesis in MPC. Cells were treated with TNF-α, IL-1β or IL-6 at a final concentration of 5 ng/ml for 18 hrs. Cells were lysed with RIPA buffer and 20 μg protein analysed by Western blotting for CSE, CBS and β-actin expression. H2S synthesis was determined by zinc-trap specrophotometry in the presence and absence of D,L-propargylglycine (PAG; to inhibit CSE) or aminooxyacetate (AOAA; to inhibit CBS) added a final concentration of 1 mmol/l as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. Figure 1(D) ***P < 0.001, cf. untreated cells; Figure 1(F) *P < 0.05, **P < 0.01, ***P < 0.001.

Mentions: Figure 1A–C shows that under basal conditions chondrogenically differentiated MPCs expressed CBS and had detectable CSE. Densitometric analysis of CSE and CBS expression is shown in Figure 1D–E, respectively. Basal levels of H2S synthesis were significantly inhibited by AOAA (a CBS inhibitor) (Fig. 1F). In contrast, a small statistically insignificant decrease in H2S synthesis was observed after treatment with the CSE inhibitor PAG, suggesting that CBS was the predominant source of H2S under basal conditions. In contrast, treatment of MPCs with the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 significantly increased expression (Fig. 1A–E) and activity (Fig. 1F) of CSE but not CBS. Incubation of chondrocytes in alginate culture with TNF-α, IL-1β and IL-6 under the same experimental conditions as MPCs also resulted in significant increases in expression (Fig. 2A and B) and activity (Fig. 2C) of CSE but not CBS. In MPCs and chondrocytes PAG, but not AOAA, significantly inhibited cytokine-induced CSE activity, suggesting that CSE is an inducible source of H2S in these cells.


Inducible hydrogen sulfide synthesis in chondrocytes and mesenchymal progenitor cells: is H2S a novel cytoprotective mediator in the inflamed joint?

Fox B, Schantz JT, Haigh R, Wood ME, Moore PK, Viner N, Spencer JP, Winyard PG, Whiteman M - J. Cell. Mol. Med. (2012)

Inducible expression and activity of CSE but not CBS in human chondrogenically differentiated mesenchymal progenitor cells (MPCs). (A–C) CSE and CBS protein expression determined by Western blotting and (D, E) Western blot analysis by densitometry. (F) Cytokine induced H2S synthesis in MPC. Cells were treated with TNF-α, IL-1β or IL-6 at a final concentration of 5 ng/ml for 18 hrs. Cells were lysed with RIPA buffer and 20 μg protein analysed by Western blotting for CSE, CBS and β-actin expression. H2S synthesis was determined by zinc-trap specrophotometry in the presence and absence of D,L-propargylglycine (PAG; to inhibit CSE) or aminooxyacetate (AOAA; to inhibit CBS) added a final concentration of 1 mmol/l as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. Figure 1(D) ***P < 0.001, cf. untreated cells; Figure 1(F) *P < 0.05, **P < 0.01, ***P < 0.001.
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fig01: Inducible expression and activity of CSE but not CBS in human chondrogenically differentiated mesenchymal progenitor cells (MPCs). (A–C) CSE and CBS protein expression determined by Western blotting and (D, E) Western blot analysis by densitometry. (F) Cytokine induced H2S synthesis in MPC. Cells were treated with TNF-α, IL-1β or IL-6 at a final concentration of 5 ng/ml for 18 hrs. Cells were lysed with RIPA buffer and 20 μg protein analysed by Western blotting for CSE, CBS and β-actin expression. H2S synthesis was determined by zinc-trap specrophotometry in the presence and absence of D,L-propargylglycine (PAG; to inhibit CSE) or aminooxyacetate (AOAA; to inhibit CBS) added a final concentration of 1 mmol/l as described in Materials and Methods. Data are expressed as mean ± S.D. of six determinations. Figure 1(D) ***P < 0.001, cf. untreated cells; Figure 1(F) *P < 0.05, **P < 0.01, ***P < 0.001.
Mentions: Figure 1A–C shows that under basal conditions chondrogenically differentiated MPCs expressed CBS and had detectable CSE. Densitometric analysis of CSE and CBS expression is shown in Figure 1D–E, respectively. Basal levels of H2S synthesis were significantly inhibited by AOAA (a CBS inhibitor) (Fig. 1F). In contrast, a small statistically insignificant decrease in H2S synthesis was observed after treatment with the CSE inhibitor PAG, suggesting that CBS was the predominant source of H2S under basal conditions. In contrast, treatment of MPCs with the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 significantly increased expression (Fig. 1A–E) and activity (Fig. 1F) of CSE but not CBS. Incubation of chondrocytes in alginate culture with TNF-α, IL-1β and IL-6 under the same experimental conditions as MPCs also resulted in significant increases in expression (Fig. 2A and B) and activity (Fig. 2C) of CSE but not CBS. In MPCs and chondrocytes PAG, but not AOAA, significantly inhibited cytokine-induced CSE activity, suggesting that CSE is an inducible source of H2S in these cells.

Bottom Line: Oxidative stress-induced cell death was significantly inhibited by GYY4137 treatment but increased by pharmacological inhibition of H(2)S synthesis or by CBS/CSE-siRNA treatment.These data suggest CSE is an inducible source of H(2)S in cultured HACs and MPCs.H(2)S may represent a novel endogenous mechanism of cytoprotection in the inflamed joint, suggesting a potential opportunity for therapeutic intervention.

View Article: PubMed Central - PubMed

Affiliation: Peninsula Medical School, University of Exeter, St. Luke's Campus, Exeter, Devon, UK.

Show MeSH
Related in: MedlinePlus