Phosphoserine-86-HSPB1 (pS86-HSPB1) is cytoplasmic and highly induced in rat myometrium at labour.

Uterine myocytes during pregnancy proceed through a series of adaptations and collectively transform into a powerfully contractile tissue by term. Previous work has indicated that members of the heat shock protein (HSP) B family of stress proteins are associated with the process of adaptation and transformation. Utilizing immunoblot analyses, widefield epifluorescence and total internal reflection (TIRF) microscopy, this study investigated the temporal and spatial detection of HSPB1 phosphorylated on serine-86 (pS86-HSPB1) in rat myometrium during pregnancy, the role of uterine distension in regulation of pS86-HSPB1, and the comparative localization with pS15-HSPB1 in rat myometrial tissue as well as in an immortalized human myometrial cell line. Immunoblot detection of pS86-HSPB1 was significantly elevated during late pregnancy and labour. In particular, pS86-HSPB1 was significantly increased at day (d)22 and d23 (labour) compared with all other timepoints assessed. Localization of pS86-HSPB1 in myometrium became prominent at d22 and d23 with cytoplasmic detection around myometrial cell nuclei. Furthermore, pS86-HSPB1 detection was found to be significantly elevated in the gravid rat uterine myometrium compared with the non-gravid tissue at d19 and d23. Both widefield epifluorescence and TIRF microscopy examination of human myometrial cells demonstrated that pS15-HSPB1 was prominently localized to focal adhesions, while pS82-HSPB1 (homologous to rodent pS86-HSPB1) was primarily located in the cell cytoplasm. Our data demonstrate that levels of phosphorylated HSPB1 increase just prior to and during labour, and that uterine distension is a stress-inducing signal for HSPB1 phosphorylation. The exact roles of these phosphorylated forms in myometrial cells remain to be determined.

Examination of FERMT1 expression in placental chorionic villi and its role in HTR8-SVneo cell invasion.

Transmembrane integrin receptors mediate cell-extracellular matrix as well as cell-cell adhesion. As placental trophoblast cells undergo differentiation they display changes in integrin expression or switching, but the mechanism(s) of integrin activation that supports this differentiation is still unknown. The Fermitin family of adapter proteins (FERMT 1-3) are integrin activators that mediate integrin-mediated signaling. In this study, we examined the spatiotemporal pattern of expression of FERMT1 in human chorionic villi throughout gestation and its role in HTR8-SVneo substrate adhesion and invasion. Placental villous tissue was obtained from patients undergoing elective terminations at weeks 8-14, as well as from term deliveries at weeks 37-40 and analyzed by immunofluorescence. Additionally, HTR8-SVneo trophoblast cells were transfected with FERMT1-specific siRNA or non-targeting siRNA (control) and used in cell-substrate adhesion as well as invasion assays. FERMT1 was primarily localized to membrane-associated regions at the base or around the periphery of the villous cytotrophoblast and proximal as well as distal cell column trophoblast. FERMT1 was also localized to endothelial cells of blood vessels in chorionic villi. siRNA-mediated depletion of FERMT1 in HTR8-SVneo cells did not markedly alter HTR8-SVneo cell-substrate adhesion but did significantly decrease invasion (P < 0.05) compared to control cells. These novel findings identify the presence of the integrin activator FERMT1 in trophoblast cells and that FERMT1 can regulate HTR8-SVneo cell invasion. FERMT1 may directly influence integrin activation and the subsequent integrin-mediated signaling and differentiation that underlies the acquisition of the invasive trophoblast phenotype in vivo.

Inducible heat shock protein A1A (HSPA1A) is markedly expressed in rat myometrium by labour and secreted via myometrial cell-derived extracellular vesicles.

The myometrium goes through physiological, cellular and molecular alterations during gestation that necessitate effective cellular proteostasis. Inducible heat shock protein A1A (HSPA1A) is a member of the 70-kDa heat shock protein A (HSPA) family, which acts as a chaperone to regulate proteostasis; however, HSPA1A also participates as a cytokine in inflammatory regulation, leading to its designation as a chaperokine. This study examined the spatiotemporal expression of HSPA1A protein in the rat myometrium throughout gestation and assessed whether it is secreted as cargo of myometrial cell-derived extracellular vesicles (EVs). Immunoblot analysis demonstrated that HSPA1A expression was markedly elevated during late pregnancy and labour and increased by uterine distension. Myometrial HSPA1A expression insitu increased in myocytes of longitudinal and circular muscle layers from Day 19 through to postpartum, specifically in the cytoplasm and nuclei of myocytes from both muscle layers, but frequently detectable just outside myocyte membranes. Scanning electron microscopy examination of samples isolated from hTERT-HM cell-conditioned culture medium, using EV isolation spin columns, confirmed the presence of EVs. EV lysates contained HSPA8, HSPA1A and the EV markers apoptosis-linked gene 2-interacting protein X (Alix), the tetraspanin cluster of differentiation 63 (CD63), tumour susceptibility gene 101 (TSG101) and HSP90, but not the endoplasmic reticulum protein calnexin. These results indicate that HSPA1A may act as a chaperokine in the myometrium during pregnancy.

The Potential Functions of Small Heat Shock Proteins in the Uterine Musculature during Pregnancy.

The small heat shock protein B (HSPB) family is comprised of eleven members with many being induced by physiological stressors. In addition to being molecular chaperones, it is clear these proteins also play important roles in cell death regulation, cytoskeletal rearrangements, and immune system activation. These processes are important for the uterine smooth muscle or myometrium during pregnancy as it changes from a quiescent tissue, during the majority of pregnancy, to a powerful and contractile tissue at labor. The initiation and progression of labor within the myometrium also appears to require an inflammatory response as it is infiltrated by immune cells and it produces pro-inflammatory mediators. This chapter summarizes current knowledge on the expression of HSPB family members in the myometrium during pregnancy and speculates on the possible roles of these proteins during myometrial programming and transformation of the myometrium into a possible immune regulatory tissue.

Induction of expression and phosphorylation of heat shock protein B5 (CRYAB) in rat myometrium during pregnancy and labour.

During pregnancy the myometrium undergoes a programme of differentiation induced by endocrine, cellular, and biophysical inputs. Small heat shock proteins (HSPs) are a family of ten (B1-B10) small-molecular-weight proteins that not only act as chaperones, but also assist in processes such as cytoskeleton rearrangements and immune system activation. Thus, it was hypothesized that HSPB5 (CRYAB) would be highly expressed in the rat myometrium during the contractile and labour phases of myometrial differentiation when such processes are prominent. Immunoblot analysis revealed that myometrial CRYAB protein expression significantly increased from day (D) 15 to D23 (labour; P<0.05). In correlation with these findings, serine 59-phosphorylated (pSer59) CRYAB protein expression significantly increased from D15 to D23, and was also elevated 1-day post-partum (P<0.05). pSer59-CRYAB was detected in the cytoplasm of myocytes within both uterine muscle layers mid- to late-pregnancy. In unilaterally pregnant rats, pSer59-CRYAB protein expression was significantly elevated in the gravid uterine horns at both D19 and D23 of gestation compared with non-gravid horns. Co-immunolocalization experiments using the hTERT-human myometrial cell line and confocal microscopy demonstrated that pSer59-CRYAB co-localized with the focal adhesion protein FERMT2 at the ends of actin filaments as well as with the exosomal marker CD63. Overall, pSer59-CRYAB is highly expressed in myometrium during late pregnancy and labour and its expression appears to be regulated by uterine distension. CRYAB may be involved in the regulation of actin filament dynamics at focal adhesions and could be secreted by exosomes as a prelude to involvement in immune activation in the myometrium.