Role of the nucleotide oligomerization domain-like receptor protein 7 in the pathology of recurrent hydatidiform moles

A molar pregnancy or hydatidiform mole (HM) is a human pregnancy with no embryo, but cystic degeneration of chorionic villi and excessive proliferation of the trophoblast. Mutations in NLRP7, a member of the nucleotide-binding domain and leucine-rich repeat-containing receptors family of proteins with roles in inflammation and apoptosis, are responsible for recurrent hydatidiform moles (RHMs). In previous studies, we demonstrated that ex vivo stimulated peripheral blood mononuclear cells (PBMCs) from patients with NLRP7 mutations secrete lower levels of interleukin 1 beta (IL1B) and tumor necrosis factor (TNF) than control cells despite the fact that patients' cells have normal to slightly higher intracellular levels of IL1B. We hypothesized that NLRP7 mutations do not alter IL1B synthesis or processing but affect the microtubules and consequently intracellular cytokines trafficking. To address this hypothesis, I first characterized the expression and subcellular localization of NLRP7 in PBMCs. Using immunofluorescence, I showed that NLRP7 is expressed in all PBMC subpopulations before and after LPS stimulation including monocytes, which are the main cells that express and secrete IL1B and TNF in response to LPS stimulation. Additionally, I found that NLRP7 localizes to the microtubule organizing center (MTOC) and the Golgi apparatus in various hematopoietic cell lines. The localization of NLRP7 to the MTOC and Golgi drew our attention to check for any possible dependence of NLRP7 signal on microtubules at these two locations. Toward this goal, I treated Epstein Barr Virus transformed B-lymphocytes (EBV) cells from control subjects with nocodazole, a microtubule disrupting agent, and found that this treatment disrupts the NLRP7 signal, which became more diffuse and fragmented around the nucleus. These results suggest that NLRP7 mutations impair cytokine secretion by affecting microtubules. The identification of KHDC3L as the second gene for RHMs prompted us to investigate the expression and subcellular localization of its protein in hematopoietic cells. Using immunofluorescence, I found that KHDC3L co-localizes with NLRP7 to the MTOC and Golgi apparatus in control EBV cells (data not shown). The similarities in the subcellular localization of NLRP7 and KHDC3L suggested that they may share a common function in IL1B and TNF secretion. To understand the role of NLRP7 and KHDC3L in RHMs, I sought to determine their subcellular localization in control human oocytes and early cleavage embryos, stages at which the disease starts. Using confocal immunofluorescence and electron microscopies, I found that NLRP7 co-localized with KHDC3L mainly to the cortical region in oocytes from the germinal vesicle (GV) until the formation of the zygote. Within the cortex, electron and high resolution confocal microscopies confirmed the co-localization of NLRP7 and KHDC3L between cortical granules, mitochondria, and other organelles on oocyte cytoskeletal structures subsequently identified as the human subcortical maternal complex (SCMC). Additionally, we found that NLRP7 co-localizes with OOEP, another member of the SCMC and depends on alpha tubulin microtubules and filamentous actin networks. Between 2-cell and morula stages, both NLRP7 and KHDC3L signals were restricted to the outer cortical regions and absent from the cell-to-cell contact region. At the blastocyst stage, NLRP7 and KHDC3L relocate to the cytoplasm and nucleus, respectively, of the inner cell mass (ICM) and the trophectoderm. Collectively, our data implicate roles for NLRP7 and KHDC3L directly or indirectly in the cytoskeleton and open up new areas of research to investigate the role of these two maternal-effect proteins in the manifestation of HM.

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