PC6 levels in uterine lavage are closely associated with uterine receptivity and significantly lower in a subgroup of women with unexplained infertility.

BACKGROUND Embryo implantation requires a healthy embryo and a receptive uterus. Uterine incompetence contributes significantly to implantation failure and infertility. To date, there are no reliable biochemical methods that can determine whether the uterus is receptive. Proprotein convertase 5/6 (PC6) is tightly regulated in the uterus and critical for receptivity and implantation; its secretory nature predicts PC6 to be secreted into the uterine cavity. The present study examines whether PC6 is detectable in uterine lavage and whether there is any correlation between secreted PC6 levels and uterine receptivity. METHODS Western blotting determined the presence of PC6 protein in uterine lavage. A sensitive and high-throughput activity assay was established and validated. This assay was applied to 103 lavages collected from different phases of the menstrual cycle from women with proven fertility or unexplained infertility. RESULTS Uterine lavage contained PC6 protein with levels paralleling enzymatic activity. PC6 levels were significantly higher in the receptive than in the non-receptive phase in fertile women, and the putative receptive phase levels in a subgroup of women with unexplained infertility were significantly lower than in the fertile counterparts. CONCLUSIONS PC6 levels in uterine lavage are significantly elevated in the luteal phase of fertile women and markedly reduced in a subgroup of women with unexplained infertility. Uterine fluid is a valuable source of material to evaluate uterine function. Detection of PC6 in uterine fluid may lead to the development of a rapid and relatively non-surgical assessment of uterine receptivity.

Dual mechanisms for the fibrate-mediated repression of proprotein convertase subtilisin/kexin type 9.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is associated with familial autosomal dominant hypercholesterolemia and is a natural inhibitor of the LDL receptor (LDLr). PCSK9 is degraded by other proprotein convertases: PC5/6A and furin. Both PCSK9 and the LDLr are up-regulated by the hypocholesterolemic statins. Thus, inhibitors or repressors of PCSK9 should amplify their beneficial effects. In the present study, we showed that PPARalpha activation counteracts PCSK9 induction by statins by repressing PCSK9 promoter activity and by increasing PC5/6A and furin expression. Quantification of mRNA and protein levels showed that various fibrates decreased PCSK9 and increased PC5/6A and furin expression. Fenofibric acid (FA) reduced PCSK9 protein content in immortalized human hepatocytes (IHH) as well as its cellular secretion. FA suppressed PCSK9 induction by statins or by the liver X receptor agonist TO901317. PCSK9 repression is occurring at the promoter level. We showed that PC5/6A and furin fibrate-mediated up-regulation is PPARalpha-dependent. As a functional test, we observed that FA increased by 30% the effect of pravastatin on the LDLr activity in vitro. In conclusion, fibrates simultaneously decreased PCSK9 expression while increasing PC5/6A and furin expression, indicating a broad action of PPARalpha activation in proprotein convertase-mediated lipid homeostasis. Moreover, this study validates the functional relevance of a combined therapy associating PCSK9 repressors and statins.

Inhibition of PC5 expression decreases CCK secretion and increases PC2 expression.

Cholecystokinin (CCK) is produced from pro CCK by a series of enzymatic cleavages. One of the enzymes thought to be important for pro CCK cleavage is prohormone convertase 5 (PC5). STC-1 cells, a mouse intestinal tumor cell line that expresses CCK, PC1, PC2, and PC5 were stably transfected with hairpin loop plasmids encoding siRNA targeting PC5 and clones were selected. CCK secretion was reduced significantly. PC5 mRNA and protein expression as measured by quantitative PCR and Western blot analysis was reduced about 50%. CCK and PC1 mRNA expression were not changed. These cells showed a three-fold increase in PC2 mRNA and protein expression. This increase may represent a compensatory mechanism triggered by the loss of PC5. The decrease in CCK in the media was due largely to loss of CCK 22. These results provide the first direct evidence that PC5 is involved in CCK processing.

Expression, purification and characterization of recombinant mouse PC6B from baculovirus-infected insect cells.

We constructed a mouse PC6B truncated mutant and introduced a tag of 6 consecutive histidines at its carboxyl terminus for simple purification. Using the baculovirus expression system and standard enzymatic assay, we obtained recombinant mouse PC6B protein and with enzymatic activity.