Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

Abstract

The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.

Fetal Membranes Contribute to Drug Transport across the Feto-Maternal Interface Utilizing the Breast Cancer Resistance Protein (BCRP)

“…The collection of placentas was approved by the Institutional Review Board (IRB) at the University of Texas Medical Branch (UTMB) at Galveston, TX (IRB approval number IRB16.0058, January 2020). The protocol allows for the collection of discarded placentas from term vaginal or term, not in labor, cesarean deliveries (TL and TNIL, respectively) from John Sealy Hospital according to our laboratory's inclusion and exclusion criteria as described in previous reports [13,14]. The protocol is exempt from subject consent as the specimens are non-identifiable and were intended to be discarded.…”

“…Our recent data showed that fetal membranes (FMs-amniochorion), along with the placenta, express various transport proteins; however, their localization within FM cells and functions have not yet been elucidated. Since human fetal membranes are involved in various protective mechanisms [11][12][13][14][15][16] during pregnancy, it is likely that transport protein functions may also be controlled by the FMs. Determining the role of FMs in drug transport is necessary and could lead to a greater understanding of pharmacokinetics in pregnancy.…”

Organic Anion Transporting Polypeptide 2B1 in Human Fetal Membranes: A Novel Gatekeeper for Drug Transport During Pregnancy?

“…Immunohistochemistry (IHC) for OATP2B1 was performed on FM tissues using methods previously described by our laboratory (Kammala et al, 2020). Briefly, FM sections were fixed in 4% paraformaldehyde for 48 h and embedded in paraffin.…”

Functional role and regulation of permeability‐glycoprotein (P‐gp) in the fetal membrane during drug transportation

“…33 Maternal decidual and FM cells were cultured as previously described. 34,28,35 The cells used for all experiments were within 10 passages.…”

“…Small interfering RNA-mediated knockdown of NHERF1 in BeWo cells and CTCs Small interfering RNA (siRNA) On-Target plus smart pool oligos (Dharmacon, Lafayette, CO, USA) directed against NHERF1 or nontarget scrambled (NT; control) siRNA was used to transfect FMs cells (AECs, AMCs, CMCs, and CTCs) using the RNAimax lipofectamine reagent as previously described. 28 Twenty-four hours after transfection, the cells were used for subsequent assessment of protein expression and stimulation.…”