Role of PCSK5 Expression in Mouse Ovarian Follicle Development: Identification of the Inhibin α- and β-Subunits as Candidate Substrates

Inhibin and activin are essential dimeric glycoproteins belonging to the transforming growth factor-beta (TGFβ) superfamily. Inhibin is a heterodimer of α- and β-subunits, whereas activin is a homodimer of β-subunits. Production of inhibin is regulated during the reproductive cycle and requires the processing of pro-ligands to produce mature hormone. Furin is a subtilisin-like proprotein convertase (proconvertase) that activates precursor proteins by cleavage at basic sites during their transit through the secretory pathway and/or at the cell surface. We hypothesized that furin-like proconvertases are central regulators of inhibin α- and β-subunit processing within the ovary. We analyzed the expression of the proconvertases furin, PCSK5, PCSK6, and PCSK7 in the developing mouse ovary by real-time quantitative RT-PCR. The data showed that proconvertase enzymes are temporally expressed in ovarian cells. With the transition from two-layer secondary to pre-antral follicle, only PCSK5 mRNA was significantly elevated. Activin A selectively enhanced expression of PCSK5 mRNA and decreased expression of furin and PCSK6 in cultured two-layer secondary follicles. Inhibition of proconvertase enzyme activity by dec-RVKR-chloromethylketone (CMK), a highly specific and potent competitive inhibitor of subtilisin-like proconvertases, significantly impeded both inhibin α- and β-subunit maturation in murine granulosa cells. Overexpression of PC5/6 in furin-deficient cells led to increased inhibin α- and βB-subunit maturation. Our data support the role of proconvertase PCSK5 in the processing of ovarian inhibin subunits during folliculogenesis and suggest that this enzyme may be an important regulator of inhibin and activin bioavailability

Modulation of GSTA1 levels mediate changes in Caco-2 cell growth.

<p>Effect of (A) GSTA1 down-regulation and (B) GSTA1-V5 overexpression on Caco-2 cell viability evaluated by MTS assay over three days. Asterisks depict significant differences between controls and the cells with GSTA1 modulated levels (*, <i>p</i>≤0.05; and **, <i>p</i>≤0.01). (C) Effect of GSTA1-V5 over-expression on cellular proliferation at 72 h as determined by BrdU incorporation in Caco-2 cells. Bars indicated by different letters differ significantly from one another (p≤0.001). Values represent the mean ± S.E. of four independent experiments with three replicates each.</p

GSTA1 over-expression does not interfere with NaB-induced apoptosis.

<p>(A) Representative western blots of V5 (∼26 KDa), endogenous caspase-3 (∼35 KDa) and activated caspase-3 (∼19 KDa and 17 KDa) in Caco-2 cells. Preconfluent Caco-2 cells were transiently transfected with one µg of either GSTA1-V5 or empty vector (EV) for 48 h and treated with NaB (10 mM) for 48 h. β-actin (∼42 KDa) was used as a protein loading control. (B) Densitometric analysis of caspase-3 activation in GSTA1 over-expressed cells with and without NaB (10 mM) treatment. Values represent the mean ± S.E of three independent experiments. Bars indicated by different letters differ significantly from one another (p≤0.001).</p

NaB (10 mM) causes GSTA1-JNK complex dissociation without activating JNK in Caco-2 cells.

<p>(A) Representative western blot of GSTA1 (∼25 KDa) and GST Pi (∼26 KDa) protein levels in GSTA1-JNK complexes. Cells were transiently transfected with GSTA1 siRNA and non-specific (NS) siRNA for 72 h and treated with 10 mM NaB. GSTA1-JNK complexes were then pulled-down from cell lysates using c-Jun fusion beads. (B) Representative western blot of phosphorylated JNK (∼54 KDa and 46 KDa) and phosphorylated p38 (∼43 KDa) protein expression in preconfluent Caco-2 cells with the treatment of 10 mM NaB. β-actin (∼42 KDa) was used as a protein loading control.</p

GSTA1 down-regulation increases the percentage of Caco-2 cells in the S phase.

<p>(A) Changes of cell cycle phase distribution in GSTA1 down-regulated Caco-2 cells as compared to controls. (B) Graphic representation of percent of cells in G1, S and G2 phase of cell cycle in non-transfected control, GSTA1 siRNA and NS siRNA transfected Caco-2 cells. Asterisks depict significant differences between control and GSTA1 down-regulated cells (*, p≤0.05; and **, p≤0.01).</p

Modulation of GSTA1 does not affect NaB-induced differentiation.

<p>Preconfluent Caco-2 cells were transiently transfected with 40 nM of GSTA1 siRNA or non-specific (NS) siRNA and after 72 h, cells were treated with 1 mM NaB for 72 h; (A) GSTA1 activity (nmol/mg/min) was determined in GSTA1 down-regulated cells. (B) AlkP activity (µmol/mg/min) was measured to determine the effect of GSTA1 down-regulation on NaB-induced differentiation. (C) Preconfluent Caco-2 cells were transiently transfected with one µg of either GSTA1-V5 or empty vector (EV) for 48 h and were treated with 1 mM NaB for 48 h. AlkP activity (µmol/mg/min) was measured to determine the effect of GSTA1 over-expression on NaB-induced differentiation. Values represent the mean ± S.E. of three independent experiments with six replicates each. Bars indicated by different letters differ significantly from one another (p≤0.001).</p

Distinct doses of NaB differently affect cell proliferation and AlkP and GSTA1 enzyme activities.

<p>Preconfluent Caco-2 cells were treated with NaB (1 mM and 10 mM) in serum-free media. (A) Cellular proliferation was assessed from 24–96 h. Asterisks depict significant differences between control and NaB treatments (*, p≤0.05; **, p≤0.01 and ***, p≤0.001). (B) Cytotoxicity was determined in preconfluent and postconfluent Caco-2 cells treated with 1 mM and 10 mM NaB at 48 h. Cytotoxicity measured LDH release and presented as % cytotoxicity. (C) AlkP activity (µmol/mg/min) and (D) GSTA1 activity (nmol/mg/min) was determined. Values represent the mean ± S.E. of three independent experiments with six replicates each. Bars indicated by different letters differ significantly from one another (p≤0.001).</p

GSTA1 down-regulation does not affect the sensitivity of Caco-2 cells to NaB-induced apoptosis.

<p>(A) Representative western blots of GSTA1 (∼25 KDa), endogenous caspase-3 (∼35 KDa), activated caspase-3 (∼19 KDa and 17 KDa) in Caco-2 cells. Preconfluent Caco-2 cells were transiently transfected with 40 nM of GSTA1 siRNA or non-specific (NS) siRNA for 72 h and treated with NaB (10 mM) for 48 h. β-actin (∼42 KDa) was used as a protein loading control. Densitometric analysis of (B) GSTA1 levels and (C) caspase-3 activation in GSTA1 down-regulated cells with and without NaB (10 mM) treatment. Values represent the mean ± S.E of three independent experiments with three replicates each. Bars indicated by different letters differ significantly from one another (p≤0.05).</p

GSTA1 levels increase in differentiating Caco-2 cells.

<p>Preconfluent and 10 d postconfluent Caco-2 cells were assessed for: (A) protein expression of GSTA1 (∼25 KDa) and GSTP1 (∼26 KDa). β-actin was used as a protein loading control; (B) GSTA1 enzyme activity (nmol/mg/min); (C) mRNA levels of differentiation markers: AlkP, villin, DPP-4 and E-cadherin by real time RT-PCR; and (D) AlkP enzyme activity (µmol/mg/min). Values represent the mean ± S.E. of three independent experiments with three replicates each. Bars indicated by different letters differ significantly from one another (p≤0.001).</p

Relative abundance and activity of GSTA1 in transiently transfected Caco-2 cells.

*<p>Each value represents the mean ± S.E of three independent experiments with three replicates each. ND, not determined; NS, non-specific. Values with different letters are significantly different from each other.</p