Role of PKCepsilon in PGF2alpha-stimulated MMP-2 secretion from human ciliary muscle cells

Abstract

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.

FIG. 1.

Effects of protein kinase C inhibitors on matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Serum-deprived human ciliary muscle cells were pretreated with vehicle, 1.0 μmol/L of chelerythrine chloride (A), or 1.0 μmol/L of Go-6976 (B) for 60 min, followed by 1.0 μmol/L of prostaglandin F2-alpha treatment for 4 h. The media were then collected, concentrated, and analyzed for MMP-2 by Western blotting, using anti-MMP-2 antibodies. Data are the mean ± standard error of densitometry measurements (n = 3–7; *P < 0.05 when compared to the control levels).

FIG. 2.

Western blot analyses for individual protein kinase C (PKC) isoforms in cytosolic (C) and membrane (M) fractions of nonstimulated human ciliary muscle cells. Equal amounts of protein (15 μg) from cytosolic or membrane fractions were subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis. Proteins were transferred to nitrocellulose membranes and probed with primary antibodies selective for each PKC isoform, as described in the Methods section.

FIG. 3.

Effects of prostaglandin F2-alpha (PGF_2α) on the translocation of protein kinase C (PKC) isoforms in human ciliary muscle cells. Serum-deprived human ciliary muscle cells were treated with vehicle or 1 μmol/L PGF_2α for 5 min, followed by the isolation of the membrane fraction and Western blot analysis. The upper panel sections are representative immunoblots of PKCα (A) and PKCɛ (B) translocations to the membrane fraction. Summary data (lower panel sections) are the mean ± standard error of densitometry (n = 3–7; *P < 0.05).

FIG. 4.

Effects of protein kinase C (PKC)ɛ translocation inhibitors on matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Serum-deprived human ciliary muscle cells were treated with vehicle or 200 nmol/L of PKCɛ translocation inhibitor, EAVSLKPT, for 60 min followed by 1 μmol/L PGF_2α treatment for 4 h. The media were then collected, concentrated, and analyzed for MMP-2 by Western blotting, using anti-MMP-2 antibodies. The upper panel section shows representative immunoblots of MMP-2. Summary data (lower panel section) are the mean ± standard error of densitometry measurements (n = 4; *P < 0.05, compared to control levels).

FIG. 5.

Inhibition of prostaglandin F2-alpha (PGF_2α)-induced matrix metalloproteinase-2 (MMP-2) secretion by siRNA targeting endogenous protein kinase C (PKC)ɛ. Human ciliary muscle cells were transfected with 10 nmol/L of siRNA targeting PKCɛ or Lamin A/C for 4 h in serum-free media. Two (2) days after transfection, cells were harvested and immunoblotted with PKC isoform and β-actin antibodies. (A) Representative immunoblots of PKCɛ and PKCα from cells treated with PKCɛ siRNA, Lamin A/C siRNA, or tranfection reagent alone (control). Beta-actin was used as an internal control to insure that equal amounts of proteins were loaded in each lane. Lamin A/C siRNA was used as a positive control for the silencing of nonessential, abundantly expressed housekeeping genes in these cells. (B) Alteration in MMP-2 secretion induced by PGF_2α (1 μmol/L) produced by siRNA transfection. The upper panel section is a representative immunoblot of MMP-2 from cells treated with PKCɛ siRNA, Lamin A/C siRNA, or tranfection reagent alone (control). Summary data (lower panel section) are the mean ± standard error of densitometry measurements (n = 3–7; *P < 0.05, compared to control levels).

FIG. 6.

Effects of protein kinase C (PKC) inhibitors on prostaglandin F2-alpha (PGF_2α)-induced ERK1/2 activation. Serum-deprived human ciliary muscle cells were pretreated with vehicle, 1 μmol/L of chelerythrine chloride or the PKCɛ translocation inhibitor, EAVSLKPT, for 60 min followed by 1 μmol/L of PGF_2α treatment for 5 min. The cell lysates were analyzed for ERK1/2 phosphorylation by Western blot, using anti-phospho-ERK1/2 and anti-ERK1/2 antibodies.