Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

Mice deficient for the fibulin-5 gene (Fbln5(-/-)) develop pelvic organ prolapse (POP) due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP)-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/-) mice, herein named V1 (25 kDa). V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS) 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/-) mice. PRSS3 was (a) localized in epithelial secretions, (b) detected in media of vaginal organ culture from both Fbln5(-/-) and wild type mice, and (c) cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin) and Elafin] was dysregulated in Fbln5(-/-) epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice

Tissue-specific expression of serine protease inhibitors in the vaginal wall.

<p><i>SERPINA1</i> (<b>A</b>) and <i>E1</i> (<b>B</b>) were expressed in vaginal stroma (<b>Str, solid bars</b>) and epithelium (<b>Epi, open bars</b>) from pre- or post-menopausal women with (<b>Prol</b>) or without (<b>Ctl</b>) pelvic organ prolapse. Serine protease inhibitors <i>Spink5</i> (<b>C</b>), <i>SLPI</i> (<b>D</b>), and <i>Elafin</i> (<b>E</b>) were predominantly expressed in vaginal epithelium. Data represent mean ± SEM of stroma samples described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056376#pone-0056376-t001" target="_blank">Table 1</a>. For epithelium: pre ctl, n = 5; pre prol, n = 8, post ctl, n = 3; post prol, n = 12. *P<0.05 compared with stroma from premenopausal controls, ANOVA, Dunnett’s post hoc testing. **P<0.01.</p

Detection of caseinolytic activity in <i>Fbln5^−/−</i> tissues.

<p>(<b>A</b>) <i>a,</i> casein zymography using vaginal extracts from wild-type (WT) and <i>Fbln5^−/−</i> (KO) at 1–2 months of age. Arrow indicates caseinolytic activity at 25-kDa. <i>b,</i> corresponding coomassie staining showing equal loading. <i>c</i>, Quantification of caseinolytic activity at 25-kDa in a. Bars are mean ± SEM. *P<0.05. (<b>B</b>) Caseinolytic activity was examined in various tissues from WT and KO mice. Note a 21-kDa band detected in vagina, skin, uterus, and prostate.</p

Expression of <i>PRSS3</i> mRNA in the human vagina.

<p>Expression of <i>PRSS</i> quantified by qPCR using primers that recognize all 3 family members (PRSS common) correlated with expression of primer-specific <i>PRSS3</i> (<b>A</b>), but not <i>PRSS2</i> (<b>B</b>). <b>C</b>. <i>PRSS3</i> mRNA levels in vaginal stroma (<b>Str, solid bars</b>) or epithelium (<b>Epi, open bars</b>) from pre- or post-menopausal women with (<b>Prol</b>) or without (<b>Ctl</b>) pelvic organ prolapse. Data represent mean ± SEM of stroma samples described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056376#pone-0056376-t001" target="_blank">Table 1</a>. For epithelium: pre ctl, n = 5; pre prol, n = 8, post ctl, n = 3; post prol, n = 12.</p

V1 shares similar biochemical properties as those of PRSS3.

<p>(<b>A</b>) Activity-based detection of serine proteases in vaginal extracts using TAMRA FP probe. PRSS3 recombinant protein was used as positive control for detecting 25 kDa serine protease activity. A band corresponding to 25 kDa along with other bands were seen in both <i>Fbln5^+/−</i> (Het) and KO vaginal extracts. V1 activity is indicated by relative density of the band. (<b>B</b>) Activity-based pull-down assay of V1 protease. Up-regulation of ∼25-kDa FP-biotin reactive band is seen in the KO vagina (lane 3, box) compared to WT (lane 2). No corresponding band was seen in lane 1, in which KO extract was incubated with streptavidin (SA) beads without FP-biotin (lane 1). V1 activity is indicated by relative density of the band. (<b>C</b>) Alignment of PRSS1-3 proteins with peptide fragments (shaded box) obtained from 25-kDa band (box in B) after mass spectrometery. (<b>D</b>) Immunoblot analysis of PRSS3 using conditioned media of vaginal organ culture from WT and KO mice. Note comparable levels of secreted immunoreactive PRSS3 in WT and KO. (<b>E</b>) Casein zymography using conditioned media described in (D). PRSS3 was used as positive control to show the caseinolytic activity of the recombinant enzyme. Note caseinolytic activity at 25 kDa (asterisk) was increased in KO conditioned media relative to that in WT. V1 activity was indicated by relative density of 25 kDa band (average of three samples). (<b>F</b>) In vitro cleavage of fibulin-5 by PRSS3. Note asterisk indicates a major cleavage product of fibulin-5. The experiment was performed three times.</p

Tissue-specific expression of select Serpins in vaginal stroma and epithelium from young (4–6 wks) and old (6–8 mo) WT and <i>Fbln5</i>^−/− (KO) mice.

<p>Epithelium was scraped from the underlying stroma of the vaginal wall from WT and KO mice. Expression of <i>Serpins a1a</i> (<b>A</b>), <i>a1b</i> (<b>B</b>), <i>a3n</i> (<b>C</b>), <i>a1c</i> (<b>D</b>), and <i>ab7</i> (<b>E</b>) was analyzed by qPCR. Epithelium from 3–4 mice was pooled for one data point. Data represent mean ± SEM of epithelium (n = 3 pools) or stroma (n = 8−10) from each genotype at each age. *P<0.05 compared with corresponding WT.</p

Compartmentalization of caseinolytic activity in <i>Fbln5^−/−</i> vagina.

<p>(<b>A</b>) Epithelium (Epi)- and stroma (St)-enriched extracts were prepared from vagina (left) and uterus (right) from <i>Fbln5^−/−</i> mice. Note strong V1 activity predominantly detected in epithelium of the vagina. An asterisk shows the activity at 21 kDa. V1 activity is indicated by relative density of the band. nd, not detected. (<b>B</b>) Effect of estrogen on V1 activity in <i>Fbln5^−/−</i> mice. Extracts were prepared from vagina of normal cycling (NC) or ovariectomized animals injected with vehicle (+Veh) or estrogen (+E2).</p

PRSS3 immunoreactivity in <i>Fbln5^−/−</i> vagina.

<p>Vaginal (a, b, d, e) or pancreas sections (c, f) from 1-month old <i>Fbln5^−/−</i> mouse were incubated with secondary antibody only (a) or anti-PRSS3 antibody (b, c) and counter-stained with DAPI (d, e, f). Note strong PRSS3 expression in vaginal secretions as well as in stroma of the <i>Fbln5^−/−</i> vagina. lu, vaginal lumen; epi, epithelium; st, stroma. Bars are 30 µm.</p

Effects of protease inhibitors on V1 activity.

<p>(<b>A</b>) Normal cycling and ovariectomized vagina extracts shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056376#pone-0056376-g002" target="_blank">Figure 2</a> were subjected to protease inhibition. EDTA (10 mM) or PMSF (100 µM) was incubated with each extract before casein zymography. Whereas EDTA showed no effect, PMSF completely inhibited V1 activity. V1 activity is indicated by relative density of the band. (<b>B</b>) <i>Fbln5^−/−</i> vaginal extracts were pre-incubated with vehicle, TLCK (5 µM), or TPCK (10 µM) for 37°C for 1 h before analyzed by casein zymography. Note that V1 activity was resistant to protease inhibitors. (<b>C</b>) Caseinolytic activity assays using EDTA (0.1–10 mM), PMSF (5–500 µM), and TLCK (5–500 µM) were incubated with <i>Fbln5^−/−</i> vaginal extracts and fluorescence intensity was measured as a caseinolytic activity. Note inhibition by PMSF and TLCK. Bars are mean ± SEM. *P<0.05, **P<0.01, and ***P<0.005 compared with KO without inhibitor.</p