Protein kinase C and human uterine contractility

At least three conventional PRKC isoforms (cPRKC) (PRKCA, PRKCB1, PRKCB2), two novel PRKC isoforms (nPRKC) (PRKCD, PRKCE) and an atypical PRKCZ isoform were detected in human pregnant myometrium [13, 14].

It seems to be less obvious whether PRKC plays a role in the kinase cascade involved in the myometrial contractile process during human pregnancy. We have attempted to individualize the PRKC isoform able to selectively elicit uterine contraction at the end of pregnancy. Selective inhibitors of cPRKC and PRKCD isoforms, do not prevent ET-1-induced contractions, whereas LY294002, a phosphoinositide-3-kinase (PI3K) inhibitor, affects the contractile response [13, 15]. These results suggest but not demonstrate a role for aPRKC isoforms in myometrial contractions. We extended the analysis to a complementary model of cultured myometrial cells, which could be utilized for identifying potential regulatory pathways that modulate normal uterine activity. In a functional assay using collagen gel retraction, we confirmed that human myometrial cells maintain a smooth muscle cell phenotype in culture and responded appropriately to the potent uterotonic agent, ET-1, via selective activation of the EDNRA receptor [16]. This technique was therefore utilized to elucidate the precise contribution of the PRKC pathways in myometrial cell contraction. As shown in Figure 2, pharmacological inhibition of cPRKC and nPRKC did not affect myometrial cell contraction. Treatment of myometrial cell lattices with an inhibitory peptide specific for PRKCZ or with an antisense-oligonucleotide (AS-ODN) against PRKCZ resulted in a significant lost of ET-1-induced contraction. In addition, activated PRKCZ associates with the actin component of the cytoskeleton of cultured human pregnant myometrial cells and may regulate the phosphorylation or binding properties of actin. These different approaches provide the first evidence that only PRKCZ activation mediated ET-1-induced myometrial contraction in late pregnancy [15]. In contrast, in one study, Ozaki et al. [17] reported that PRKC activation by phorbol ester possibly through PRKCB pathway, enhances contraction induced by high K+ in the pregnant human myometrium with increasing Ca2+ sensitivity of contractile elements, while a report by Bermeo et al. [18] found that magnesium sulfate and oxytocin require extracellular calcium to induce activation of both PRKCA and PRKCD in cultured myometrial cells from pregnant women. The controversial role of PRKC isoforms in uterine contractility required clarification.

The control of the onset of labor in women is as yet unknown, but the implication of the PRKCZ in the contractile uterine process at the time of parturition is not so surprising.

Why? Modulation of the immune/inflammatory mechanisms, specially a balance between T helper (Th1)/Th2 cytokine production, has been described at the time of parturition, and a link between TNF/IL1B and premature human childbirth has been proposed [19]. Additionally, aPRKCs are important components of the TNF/IL1B signaling pathway that control NFKB activation [20‐22]. Undoubtedly, it is important to elucidate the probable interaction between these aPRKC isoforms and the cascade of pro-inflammatory cytokines.

Approximately 20% to 33% of preterm birth is associated with an underlying infection process. Systemic or local intrauterine infections have been implicated in the pathogenesis of preterm labor and delivery (for a review, see [23]). Exposure to microbial insult induces preterm birth in several animal models. For example in the pregnant mouse, the endotoxin lipopolysaccharide (LPS) (a glycolipid derived from the outer membrane of gram-negative bacteria)-enhances amplitude of in vitro spontaneous uterine contractile activity [24] and premature labor and delivery can be induced in vivo by both local and systemic administration of LPS [25‐27].

In humans, bacterial vaginosis and intrauterine infection with Gram-negative are now believed to be an important factor of risk for preterm labor [28]. The invasion of microorganisms in the utero-genital sphere triggers a release of pro-inflammatory cytokines such as TNF/IL1B. Activation of the cytokine cascade results in the production of uterotonic agents leading to parturition. Resident macrophages are thought to be the principal cell type in the female genital tract with a receptor-mediated signal transduction mechanism that is essential for immune responses to LPS [29].

Further analysis has been conducted on a model of bacterial LPS-induced inflammation of myometrial cells in order to examine a potential role of PRKCZ in this process. This in vitro model using LPS should mimic human infection-associated preterm labor. Myometrial cells express TLR4 receptors and have the potential to respond to LPS. This is further supported by the activation of PRKCZ by LPS and by the fact that LPS is able to activate the NFKB pathway. As shown in Figure 3, incubation of human myometrial cells with AS-ODN to PRKCZ abolished the LPS-induced nuclear translocation of NFKB p65 subunit [30]. It is therefore conceivable that NFKB p65 signalling pathway includes PRKCZ in our cell model. Unraveling the whole cascade of NFKB activation occurring in myometrial cells remains a difficult challenge. The enigma becomes complex, because of a switch in NFKB subunit composition in the human myometrium during pregnancy and labor [31]. In addition, increased expression of NFKB in the human myometrium at end of pregnancy is involved in functional progesterone withdrawal [32]. A question arising from these observations is whether activated NFKB also increases expression of target genes that cause increased myometrial contractility. One plausible candidate is the cyclooxygenase-2 (also known as prostaglandin-endoperoxide synthase 2, PTGS2) that might be included among the members of the "cassette of contraction-associated proteins". Recently, Duggan et al. [33] have established that aPRKCs mediate NFKB transcriptional activation and form part of the signaling network required for IL1B-induced PTGS2 expression in human cultured myometrial cells.