Hydrogen sulfide (H2S) is endogenously generated by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). It acts as a gaseous mediator in multiple signaling pathways including those involved in bone metabolism. Previous studies indicate that its level in human body is critical to maintain bone homeostasis, so that its significant decrease was observed in post-menopausal osteoporosis. Since CSE was recently found to majorly contribute to endogenous H2S production in primary osteoblasts, we are interested to study the regulation of its expression investigating CSE expression in different cell culture conditions and the activity of specific regulatory gene promoter regions. In particular, we focused on the issue regarding a gender-related H2S biosynthesis since, by using different experimental models, recent evidences support the interaction of CSE/H2S pathway with estrogen or androgen in specific districts including vascular and bone tissue. For this study we used human primary osteoblasts (hOBs) obtained from male and female vertebral bone: they express comparable levels of CSE protein as demonstrated by Western blot analysis. Bioinformatic analysis performed on the CSE gene promoter revealed the presence of putative Estrogen Responsive Elements, in addition to the Sp1 binding sites previously described. In order to analyze the potential involvement of Estrogen Receptor alpha (ER) and SP1 in the CSE gene expression, a fragment of CSE genomic region (-592/+139), wich represents the core promoter, was cloned into the upstream of firefly luciferase gene in the pGL3-Basic vector (pGL3-731). The construct was transiently transfected into hOBs under different conditions. Measuring luciferase activity by Luc assay we showed that the (-592/+139) construct presents similar activity respect to pGL3-Basic in all hOBs analyzed, but was differently affected by co-transfection with ER and Sp1 expression vectors. Specifically, in all hOBs, exogenous Sp1 overexpression as well as estrogen exposure did not significantly alter the luciferase reporter activity. On the contrary, ER overexpression together 10 nM 17-estradiol treatment deeply increased promoter activity in female hOBs but not in male hOBs. In the next step, the functional significance of ER and Sp1 on CSE gene regulation was investigated by “in vivo” chromatin immunoprecipitation (ChIP) assay. For this purpose male and female hOBs were properly cultured and collected for ChIP assay using antibodies against hERα and hSp1, and combined with semi-quantitative PCR detection. ChIP analyses revealed that both Sp1 and ER were recruited into the proximal region of CSE promoter. However, the ER recruitment was stronger in female hOBs than in male hOBs. In conclusion, we demonstrated, for the first time, that Estrogen Receptor alpha transcription factor regulates CSE promoter activity and is recruited at specific regulatory sites in hOBs. We are now investigating i. the possibility that male and female hOBs differ in production of co-regulators of estrogen-mediated CSE transcription regulation, and ii. whether also in osteoprogenitors (such as mesenchymal stem cells, MSCs) CSE expression is affected by this estrogen dependent regulation. Even though preliminary, our results results open an interesting scenario for evaluating the sexual hormones impact on H2S production and on functional activity of CSE enzyme in the bone physiopathological context.

The regulation of cystathionine γ-lyase (CSE) gene expression in human primary osteoblasts from vertebral bone: a matter of gender?

Lambertini e
Primo
Conceptualization
;
Penolazzi L
Secondo
Conceptualization
;
Angelozzi M
Methodology
;
LISIGNOLI, GINA
Methodology
;
de bonis P
Supervision
;
M cavallo
Penultimo
Supervision
;
R Piva
Ultimo
Conceptualization
2017

Abstract

Hydrogen sulfide (H2S) is endogenously generated by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE). It acts as a gaseous mediator in multiple signaling pathways including those involved in bone metabolism. Previous studies indicate that its level in human body is critical to maintain bone homeostasis, so that its significant decrease was observed in post-menopausal osteoporosis. Since CSE was recently found to majorly contribute to endogenous H2S production in primary osteoblasts, we are interested to study the regulation of its expression investigating CSE expression in different cell culture conditions and the activity of specific regulatory gene promoter regions. In particular, we focused on the issue regarding a gender-related H2S biosynthesis since, by using different experimental models, recent evidences support the interaction of CSE/H2S pathway with estrogen or androgen in specific districts including vascular and bone tissue. For this study we used human primary osteoblasts (hOBs) obtained from male and female vertebral bone: they express comparable levels of CSE protein as demonstrated by Western blot analysis. Bioinformatic analysis performed on the CSE gene promoter revealed the presence of putative Estrogen Responsive Elements, in addition to the Sp1 binding sites previously described. In order to analyze the potential involvement of Estrogen Receptor alpha (ER) and SP1 in the CSE gene expression, a fragment of CSE genomic region (-592/+139), wich represents the core promoter, was cloned into the upstream of firefly luciferase gene in the pGL3-Basic vector (pGL3-731). The construct was transiently transfected into hOBs under different conditions. Measuring luciferase activity by Luc assay we showed that the (-592/+139) construct presents similar activity respect to pGL3-Basic in all hOBs analyzed, but was differently affected by co-transfection with ER and Sp1 expression vectors. Specifically, in all hOBs, exogenous Sp1 overexpression as well as estrogen exposure did not significantly alter the luciferase reporter activity. On the contrary, ER overexpression together 10 nM 17-estradiol treatment deeply increased promoter activity in female hOBs but not in male hOBs. In the next step, the functional significance of ER and Sp1 on CSE gene regulation was investigated by “in vivo” chromatin immunoprecipitation (ChIP) assay. For this purpose male and female hOBs were properly cultured and collected for ChIP assay using antibodies against hERα and hSp1, and combined with semi-quantitative PCR detection. ChIP analyses revealed that both Sp1 and ER were recruited into the proximal region of CSE promoter. However, the ER recruitment was stronger in female hOBs than in male hOBs. In conclusion, we demonstrated, for the first time, that Estrogen Receptor alpha transcription factor regulates CSE promoter activity and is recruited at specific regulatory sites in hOBs. We are now investigating i. the possibility that male and female hOBs differ in production of co-regulators of estrogen-mediated CSE transcription regulation, and ii. whether also in osteoprogenitors (such as mesenchymal stem cells, MSCs) CSE expression is affected by this estrogen dependent regulation. Even though preliminary, our results results open an interesting scenario for evaluating the sexual hormones impact on H2S production and on functional activity of CSE enzyme in the bone physiopathological context.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11392/2381373
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