Molecular Modelling of Islet β-Cell Adaptation to Inflammation in Pregnancy and Gestational Diabetes Mellitus

Gestational diabetes mellitus (GDM), a metabolic disease that develops with the increase in insulin resistance during late pregnancy, is currently one of the most common complications affecting pregnancy. The polygenic nature of GDM, together with the interplay between different genetic variants with nutritional and environmental factors has hindered the full understanding of the etiology of this disease. However, an important genetic overlap has been found with type 2 diabetes mellitus (T2DM) and, as in the case of T2DM, most of the identified loci are associated with β-cell function. Early detection of GDM and adequate interventions to control the maternal glycemia are necessary to avoid the adverse outcomes for both the mother and the offspring. The in utero exposure to the diabetic milieu predispose these children for future diseases, among them T2DM, originating a vicious circle implicated in the increased prevalence of both GDM and T2DM. The involvement of inflammatory processes in the development of GDM highlights the importance of pancreatic β-cell factors able to favor the adaptation processes required during gestation, concomitantly with the protection of the islets from an inflammatory milieu. In this regard, two members of the Pax family of transcription factors, PAX4 and PAX8, together with the chromatin remodeler factor HMG20A, have gained great relevance due to their involvement in β-cell mass adaptation together with their anti-inflammatory properties. Mutations in these factors have been associated with GDM, highlighting these as novel candidates for genetic screening analysis in the identification of women at risk of developing GDM.

Systematic alanine scanning of PAX8 paired domain reveals functional importance of the N-subdomain

Thyroid-specific transcription factor PAX8 has an indispensable role in the thyroid gland development, which is evidenced by the facts thatPAX8/Pax8mutations cause congenital hypothyroidism in humans and mice. More than 90% of knownPAX8mutations were located in the paired domain, suggesting the central role of the domain in exerting the molecular function. Structure-function relationships of PAX8, as well as other PAX family transcription factors, have never been investigated in a systematic manner. Here, we conducted the first alanine scanning mutagenesis study, in which 132 alanine variants located in the paired domain of PAX8 were created and systematically evaluatedin vitro. We found that 76 alanine variants (55%) were loss of function (LOF) variants (defined by <30% activity as compared with wild type PAX8). Importantly, the distribution of LOF variants were skewed, with more frequently observed in the N-subdomain (65% of the alanine variants in the N-subdomain) than in the C-subdomain (45%). Twelve out of 13 alanine variants in residues that have been affected in patients with congenital hypothyroidism were actually LOF, suggesting that the alanine scanning data can be used to evaluate the functional importance of mutated residues. Using ourin vitrodata, we tested the accuracy of seven computational algorithms for pathogenicity prediction, showing that they are sensitive but not specific to evaluate on the paired domain alanine variants. Collectively, our experiment-based data would help better understand the structure-function relationships of the paired domain, and would provide a unique resource for pathogenicity prediction of futurePAX8variants.

Involvement of pax2 in ovarian development and recrudescence of catfish: a role in steroidogenesis

PAX2, a member of paired box family, is an essential transcription factor for the organ development in vertebrates including teleosts, yet no evidence has been shown for its involvement in reproduction. To study this, partial- and/or full-length cDNA of pax2 was isolated from the ovary of catfish, Clarias batrachus, along with its other Pax family members, pax1 and pax9. Tissue distribution and ontogeny expression analysis indicated the prevalence of pax2 but not pax1 and pax9 in ovary. Varied phase-wise expression during ovarian cycle and elevation of pax2 after human chorionic gonadotropin induction showed probable regulation by gonadotropins. Pax2 could be localized in various stages of oocytes and in follicular layer of vitellogenic and post-vitellogenic oocytes. To assess the functional significance of pax2, transient RNA silencing was performed using primary catfish ovarian follicle culture, in vitro, and in catfish, in vivo, through ovary-targeted injection of PEI-esiRNA. Pax2 siRNA treatment reduced the expression of various transcripts related to ovarian development like signaling molecules such as wnt4 and wnt5, estrogen receptors, several steroidogenic enzymes and transcription factors. These transitions in transcript levels might have been mediated by Pax2 acting upstream of wnt4/5 that may play a role in steroidogenesis and/or ovarian development along with ad4bp/sf-1 or by direct or indirect interaction with steroidogenic enzyme genes, which is evident from the change in the levels of serum estradiol-17β but not 17α,20β-dihydroxy-4-pregnen-3-one. Taken together, it seems that pax2 has a plausible role during ovarian development and/or recrudescence of catfish either directly or indirectly through Wnt signaling pathway.

Co-Opting the Genetic Redundancy of the Pax Family to Treat PAX5 Mutated Pre-B ALL

Pre-B cell ALL is the most common pediatric malignancy. More than a third of cases acquire loss-of-function mutations of PAX5, a regulatory transcription factor which is required for the differentiation of B lymphocytes. Additionally, germline loss of function PAX5 mutation is a cause of familial pre-B cell ALL. These observations indicate that loss of PAX5 activity drives leukemogenesis. We have seen that PAX5 re-expression in PAX5 -mutated pre-B cell ALL restores differentiation capacity and promotes apoptosis. However, because the gene is deleted or otherwise mutated it may not be possible to easily repair or restore PAX5 expression in leukemic cells. Nevertheless, PAX5 possesses two closely related paralogs, PAX2 and PAX8, which are structurally and functionally similar to PAX5, yet are neither expressed in lymphocytes nor mutated in ALL. Toward the goal of exploiting PAX family genomic redundancy for therapeutic purposes, we have tested the ability of PAX2 and PAX8 to replace PAX5 in pre-B ALL cell lines. Preliminary results indicate that both PAX2 and PAX8 rescue B cell differentiation and function similarly to PAX5 in this context. These results include modulation of B cell developmental markers, CD10, CD19 and CD22, as well as induction of PAX5 target genes, CD79a and BACH2. Cells also display a reduction in size which may be indicative of the large to small B cell transition and importantly show increased levels of cell death following PAX2 and PAX8 expression. We have further investigated the epigenetic status of PAX2 and PAX8 promoters in B cells and find that both loci are hypermethylated, suggesting that demethylation with agents such as methotrexate, may represent a therapeutic entry point for activating expression of PAX5 paralogs. Finally, based on observations that electrical polarization during early embryogenesis regulates developmental transcription factor expression, including for PAX genes, we have tested the ability of approved drugs targeting ion channels for their ability to induce PAX factor expression and thereby complement for loss of PAX5 in pre-B cell ALL. The anti-parasitic drug ivermectin, which activates chloride channels, is one of several compounds that appear promising in early studies. Disclosures No relevant conflicts of interest to declare.

Pair box (PAX8) protein to predict disease recurrence and its association with outcome in women with endometrial cancer: A retrospective study of 229 patients.

5091 Background: Pax-8 is a member of the pair-box (PAX) family of transcription factor genes and it has been found to be overexpressed in numerous cancer cell lines including ovarian and endometrial. Possibly, inhibition of Pax8 activity may even have an impact on cancer treatment. However the role of Pax8 in human endometrial cancer has not yet been explored. Thus, the aim of this study is to determine its predictive value in disease outcome of endometrial cancer. Methods: 229 patients with available clinical data and paraffin-embedded tissue were available for review and analysis. The clinical parameters used for modeling were age, histologic subtypes, myometrial depth of invasion, lympho-vascular invasion (LVI), FIGO grade, lymph nodes positive, recurrence, disease status, recurrence time and survival time. To test the association between Pax8 and the clinical parameters, Fisher’s exact test was performed. For survival analysis, Kaplan-Meier method was performed. Results: We found a strong association between PAX8+ and high tumor grade (p=0.002), LVI + (p<0.018), and type II tumor subtype. Patients with tumor expressing Pax8 were more likely to present with shorter OS and DFS p= 0.00096 and p=0.018 respectively. There was an association of PAX8 with OS (p=0.01486) with 5-years OS probability of 80.04% for patients with Pax8- and 55.9% for patients with Pax8+. There was also an association of PAX8 and DFS probability (p=0.02028) with 5-years DFS probability was of 72.12% for patients with Pax8- versus 49.88% for patients with Pax8+ expression. Conclusions: Pax8 is a reliable marker in endometrial cancer and its overexpression can predict poor outcome.