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. 2015 Jul;27(4):140-5.
doi: 10.1038/ijir.2015.5. Epub 2015 Apr 2.

Opiorphin-dependent upregulation of CD73 (a key enzyme in the adenosine signaling pathway) in corporal smooth muscle cells exposed to hypoxic conditions and in corporal tissue in pre-priapic sickle cell mice

S Fu  1 , K P Davies  2
Affiliations

Opiorphin-dependent upregulation of CD73 (a key enzyme in the adenosine signaling pathway) in corporal smooth muscle cells exposed to hypoxic conditions and in corporal tissue in pre-priapic sickle cell mice

S Fu et al. Int J Impot Res. 2015 Jul.

Abstract

The precise molecular mechanisms underlying priapism associated with sickle cell disease remain to be defined. However, there is increasing evidence that upregulated activity of the opiorphin and adenosine pathways in corporal tissue, resulting in heighted relaxation of smooth muscle, have an important role in development of priapism. A key enzyme in the adenosine pathway is CD73, an ecto-5'-nucleotidase (5'-ribonucleotide phosphohydrolase; EC 3.1.3.5) which catalyzes the conversion of adenosine mononucleotides to adenosine. In the present study we investigated how sickle cell disease and hypoxia regulate the interplay between opiorphin and CD73. In the corpora of sickle cell mice we observed significantly elevated expression of both the mouse opiorphin homolog mSmr3a (14-fold) and CD73 (2.2-fold) relative to non-sickle cell controls at a life stage before the exhibition of priapism. Sickle cell disease has a pronounced hypoxic component, therefore we determined if CD73 was also modulated in in vitro corporal smooth muscle (CSM) models of hypoxia. Hypoxia significantly increased CD73 protein and mRNA expression by 1.5-fold and 2-fold, respectively. We previously demonstrated that expression of another component of the adenosine signaling pathway, the adensosine 2B receptor, can be regulated by sialorphin (the rat opiorphin homolologue), and we demonstrate that sialorphin also regulates CD73 expression in a dose- and time-dependent fashion. Using siRNA to knockdown sialorphin mRNA expression in CSM cells in vitro, we demonstrate that the hypoxic upregulation of CD73 is dependent on the upregulation of sialorphin. Overall, our data provide further evidence to support a role for opiorphin in CSM in regulating the cellular response to hypoxia or sickle cell disease by activating smooth muscle relaxant pathways.

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Figures

Figure 1
Figure 1. (A) Upregulation of genes encoding the mouse opiorphin homologues and CD73 in corporal tissue of sickle cell mice
Expression profiles of mSmr3a, mSmr2, and CD73 in corporal tissue in control and sickle mice at the age of 5 weeks and 12 weeks, normalized to the house-keeping gene rpl19. Bars represent the mean change in gene expression in sickle cell mice compared to controls. 5 animals were used in each age group. *=P<0.05 significantly increased fold-change in gene expression compared to non-sickle cell control. (B) A representative immunoblot to analyze protein expression of CD73 in CSM cells incubated for 24 hours in low oxygen tension (1% O2, 5% CO2, and 94% N2, hypoxia) or normoxic conditions (20% O2, control). (C) Densitometric analysis was performed from three separate experiments to determine expression of CD73 and GAPDH. Expression of CD73 was normalized to the housekeeping gene GAPDH. The mean fold-change (± Std. Dev.) in expression of CD73 caused by hypoxia relative to the non-hypoxic controls are shown. *=P<0.05 significantly increased fold-change in protein expression compared to control. (D) A representative immunoblot is shown for analysisof CD73 protein expression in rat CSM cells with or without 100 μM CoCl2 incubation for 24 hours. (E) Densitometric analysis was performed from three separate experiments to determine expression of CD73 and GAPDH. Expression of CD73 was normalized to the housekeeping gene GAPDH. The mean fold-change (± Std. Dev.) in expression of CD73 caused by CoCl2-induced hypoxia relative to the non-hypoxic controls are shown. *=P<0.05 significantly increased fold-change in protein expression compared to control.
Figure 2
Figure 2. Sialorphin regulates expression of CD73 in a dose and time-dependent manner
(A, lower panel) A representative immunoblot to analyze CD73 protein expression in rat CSM after 8 hours incubation with increasing concentrations of sialorphin. (A, upper panel) Densitometry analysis of ratio of CD73 to GAPDH protein expression with increasing sialorphin concentrations. Densitometric analysis was performed from three separate experiments to determine expression of CD73 and GAPDH. Expression of CD73 was normalized to the housekeeping gene GAPDH. The mean fold-change (± Std. Dev.) in expression of CD73 caused by increasing sialorphin concentration relative to untreated controls are shown. *=P<0.05 significantly increased fold-change in protein expression compared to untreated control. (B, lower panel) A representative immunoblot to analyze time dependent protein expression ofCd73 in rat corporal cells with 400 nM sialorphin. (B, upper panel) Densitometric analysis was performed from three separate experiments to determine expression of CD73 and GAPDH. Expression of CD73 was normalized to the housekeeping gene GAPDH. The mean fold-change (± Std. Dev.) in expression of CD73 at different time points, compared to protein expression at baseline (T=0), are shown. *=P<0.05 significantly increased fold-change in protein expression compared to baseline.
Figure 3
Figure 3. Up regulation of CD73 expression by hypoxia is dependent on sialorphin
(A) In preliminary experiments CSM cells were treated for 48 hours with different concentrations of vcsa1-siRNA for 48 hours and expression of vcsa1 determined by quantitative-RT-PCR and expressed as fold change relative to the untreated cells. All concentrations gave a significant decrease in vcsa1 expression (*= P<0.05). (B) CSM cells were pretreated for 48 hours with 10 nM control-siRNA or 10 nM vcsa1-siRNA, and cells were then exposed for 8 hours to CoCl2 to mimic hypoxia. Gene expression of CD73 and rpl24 was determined in the presence or absence of CoCl2, in CSM cells pre-treated with control-siRNA or vcsa1-siRNA. Three separate experiments were performed with each gene being measured in triplicate. Data were normalized to the house keeping gene, rpl24, and expressed relative to the non-hypoxic CSM cells treated with control-siRNA. Data is expressed as mean ± Std. Dev. *=P<0.05 significantly increased, v= P<0.05 significantly decreased fold-change in gene expression compared to control (non-hypoxic CSM cells treated with control-siRNA). (C, lower panel) A representative immunoblot for the expression of CD73 and GAPDH. (C, upper panel) Densitometric analysis was performed from three separate experiments to determine expression of CD73 and GAPDH. Expression of CD73 was normalized to the housekeeping gene, GAPDH. The mean fold-change (± Std. Dev.) in expression of CD73 was compared between cells in hypoxic conditions with non-hypoxic cells for each of the treatment conditions *=P<0.05 significantly increased fold-change in protein expression compared to non-hypoxic controls.
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