|dc.description.abstract||During pregnancy the myometrium undergoes a program of differentiation in parallel with immune system transformation. The end result is the production of a powerfully contractile tissue able to deliver a term fetus. Small heat shock proteins (sHSPs) are a family of 10 proteins (HSPB1-10) involved in regulating stress responses in tissues, including inflammation, and may take part in intercellular communication via extracellular vesicles (EVs). Since HSPB1 and HSPB5 are highly expressed in myometrium during late pregnancy and labour, it was hypothesized that HSPB1 and HSPB5 expression would be induced in myometrial cells by a pro- inflammatory stimulus, in correlation with the Nuclear Factor kappa B (NF-kB) signaling pathway. Based on the literature, it was also hypothesized that these proteins would be released extracellularly in EVs. Immortalized human myometrial cells (hTERT-HM) were incubated with 1 ng/ml of IL-1β or vehicle for 0.5, 1, 3, or 6 hours (h). The expression of sHSPs and markers of inflammation in these cells were then assessed by immunoblot analysis. Detection of pSer82- HSPB1 and pSer59-HSPB5 proteins was significantly elevated in cells at 0.5 h of cytokine incubation compared to vehicle control. Activation of NF-kB, marked by phosphorylation on serine-536, was also significantly elevated after 0.5 h. In contrast, inhibitor of kappa B (IkB) expression was significantly decreased after 1 h of cytokine incubation. Thus, pSer82-HSPB1 and pSer59-HSPB5 levels correlate with NF-kB activation. Analysis of cell conditioned culture media in this experimental model also demonstrated myometrial cell secretion of EVs. Scanning electron microscopy and transmission electron microscopy verified EVs as round vesicles of 20 to 200 nm in diameter regardless of whether or not cells were exposed to IL-1β. Immunoblot analysis demonstrated EVs contained the markers cluster of differentiation 63 (CD63), apoptosis linked gene 2-interacting protein X (ALIX), and tumor susceptibility gene 101 (TSG101).
Furthermore, HSP90, HSP70, HSPB1, and NF-kB were all detected in EVs as cargo for extracellular release. These results suggest a novel mechanism of myometrial cell intercellular
communication during pregnancy that could help modulate the pro-inflammatory response in the myometrium at labour.||