Mice with a Pax2 missense variant display impaired glomerular repair.

PAX2 regulates kidney development, and its expression persists in parietal epithelial cells (PECs), potentially serving as a podocyte reserve. We hypothesized that mice with a Pax2 pathogenic missense variant (Pax2A220G/+) have impaired PEC-mediated podocyte regeneration. Embryonic wild-type mouse kidneys showed overlapping expression of PAX2/Wilms' tumor-1 (WT-1) until PEC and podocyte differentiation, reflecting a close lineage relationship. Embryonic and adult Pax2A220G/+ mice have reduced nephron number but demonstrated no glomerular disease under baseline conditions. Pax2A220G/+ mice compared with wild-type mice were more susceptible to glomerular disease after adriamycin (ADR)-induced podocyte injury, as demonstrated by worsened glomerular scarring, increased podocyte foot process effacement, and podocyte loss. There was a decrease in PAX2-expressing PECs in wild-type mice after adriamycin injury accompanied by the occurrence of PAX2/WT-1-coexpressing glomerular tuft cells. In contrast, Pax2A220G/+ mice showed no changes in the numbers of PAX2-expressing PECs after adriamycin injury, associated with fewer PAX2/WT-1-coexpressing glomerular tuft cells compared with injured wild-type mice. A subset of PAX2-expressing glomerular tuft cells after adriamycin injury was increased in Pax2A220G/+ mice, suggesting a pathological process given the worse outcomes observed in this group. Finally, Pax2A220G/+ mice have increased numbers of glomerular tuft cells expressing Ki-67 and cleaved caspase-3 compared with wild-type mice after adriamycin injury, consistent with maladaptive responses to podocyte loss. Collectively, our results suggest that decreased glomerular numbers in Pax2A220G/+ mice are likely compounded with the inability of their mutated PECs to regenerate podocyte loss, and together these two mechanisms drive the worsened focal segmental glomerular sclerosis phenotype in these mice.NEW & NOTEWORTHY Congenital anomalies of the kidney and urinary tract comprise some of the leading causes of kidney failure in children, but our previous study showed that one of its genetic causes, PAX2, is also associated with adult-onset focal segmental glomerular sclerosis. Using a clinically relevant model, our present study demonstrated that after podocyte injury, parietal epithelial cells expressing PAX2 are deployed into the glomerular tuft to assist in repair in wild-type mice, but this mechanism is impaired in Pax2A220G/+ mice.

PAX2 is regulated by estrogen/progesterone through promoter methylation in endometrioid adenocarcinoma and has an important role in carcinogenesis via the AKT/mTOR signaling pathway.

Endometrioid adenocarcinoma (EEC) is one of the most common cancers of the female reproductive system. In recent years, much emphasis has been placed on early diagnosis and treatment. PAX2 (Paired box 2) inactivation is reportedly an important biomarker for endometrioid intraepithelial neoplasia (EIN) and EEC. However, the role of PAX2 in EEC carcinogenesis remains unclear. PAX2 expression and associated clinical characteristics were analyzed via The Cancer Genome Atlas, Gene Expression Omnibus, and Cancer Cell Line Encyclopedia databases and clinical paired EIN/EEC tissue samples. Bioinformatic analysis was conducted to identify the putative molecular function and mechanism of PAX2. Cell proliferation, colony formation, cell migration, and invasion assays in vitro, and mouse xenograft models were utilized to study the biological functions of PAX2 in vivo. Pyrosequencing and the demethylating drug 5-Aza-dc were used to verify promoter methylation in clinical tissues and cell lines, respectively. The mechanism underlying the regulatory effect of estrogen (E2) and progesterone (P4) on PAX2 expression was investigated by receptor block assay and double luciferase reporter assay. PAX2 expression was found to be significantly downregulated in EIN and EEC tissues, its overexpression inhibited EEC cell malignant behaviors in vivo and in vitro and inhibited the AKT/mTOR signaling pathway. PAX2 inactivation in EEC was related to promoter methylation, and its expression was regulated by E2 and P4 through their receptors via promoter methylation. Our findings elucidated the expression and function of PAX2 in EEC and have provided hitherto undocumented evidence of the underlying molecular mechanisms. PAX2 expression is suppressed by estrogen prompting its methylation through estrogen receptor. Furthermore, PAX2 regulates the AKT/mTOR signaling pathway to influence EEC progression. © 2024 The Pathological Society of Great Britain and Ireland.

Pax protein depletion in proximal tubules triggers conserved mechanisms of resistance to acute ischemic kidney injury preventing transition to chronic kidney disease.

Acute kidney injury (AKI) is a common condition that lacks effective treatments. In part, this shortcoming is due to an incomplete understanding of the genetic mechanisms that control pathogenesis and recovery. Identifying the molecular and genetic regulators unique to nephron segments that dictate vulnerability to injury and regenerative potential could lead to new therapeutic targets to treat ischemic kidney injury. Pax2 and Pax8 are homologous transcription factors with overlapping functions that are critical for kidney development and are re-activated in AKI. Here, we examined the role of Pax2 and Pax8 in recovery from ischemic AKI and found them upregulated after severe AKI and correlated with chronic injury. Surprisingly, proximal-tubule-selective deletion of Pax2 and Pax8 resulted in a less severe chronic injury phenotype. This effect was mediated by protection against the acute insult, similar to pre-conditioning. Prior to injury, Pax2 and Pax8 mutant mice develop a unique subpopulation of proximal tubule cells in the S3 segment that displayed features usually seen only in acute or chronic injury. The expression signature of these cells was strongly enriched with genes associated with other mechanisms of protection against ischemic AKI including caloric restriction, hypoxic pre-conditioning, and female sex. Thus, our results identified a novel role for Pax2 and Pax8 in mature proximal tubules that regulates critical genes and pathways involved in both the injury response and protection from ischemic AKI.

Dihydromyricetin protects against gentamicin-induced nephrotoxicity via upregulation of renal SIRT3 and PAX2.

AIM: Gentamicin-induced nephrotoxicity limits its widespread use as an effective antibacterial agent. Oxidative stress, inflammatory cytokines and apoptotic cell death are major participants in gentamicin-induced nephrotoxicity. We therefore, investigated whether dihydromyricetin (DHM), the antioxidant and anti-inflammatory flavonoid, could protect against the nephrotoxic effects of gentamicin. METHODS: Male Wistar rats administrated gentamicin (100 mg/kg/day, i.p.) for 8 days. DHM (400 mg/kg, p.o.) was concurrently given with gentamicin for 8 days. Control group received the vehicle of DHM and gentamicin. Histopathological examinations, biochemical measurements and immunohistochemical analyses were done at the end of the study. KEY FINDINGS: Treatment with DHM improved the gentamicin induced deterioration of renal functions; serum levels of urea, creatinine and cystatin-C as well as urinary levels of Kim-1 and NGAL, the sensitive indicators for early renal damage, were declined. Additionally, DHM abrogated gentamicin-induced changes in kidney morphology. These nephroprotective effects were possibly mediated via decreasing renal gentamicin buildup, activating the antioxidant enzymes GSH, SOD and CAT and decreasing lipid peroxidation and nitric oxide levels. Further, DHM suppressed renal inflammation and apoptotic cell death by decreasing the expression of nuclear factor-kappa B (NF-κB), TNF-alpha and caspase-3. These effects were correlated to the upregulation of renal SIRT3 expression. Also, DHM activated the regeneration and replacement of injured tubular cells with new ones via enhancing PAX2 expression. SIGNIFICANCE: DHM is a promising therapeutic target that could prevent acute renal injury induced by gentamicin and help renal tubular cells to recover through its antioxidant, anti-inflammatory and antiapoptotic properties.

Co-Expression of Multiple PAX Genes in Renal Cell Carcinoma (RCC) and Correlation of High PAX Expression with Favorable Clinical Outcome in RCC Patients.

Renal cell carcinoma (RCC) is the most common form of kidney cancer, consisting of multiple distinct subtypes. RCC has the highest mortality rate amongst the urogenital cancers, with kidney renal clear cell carcinoma (KIRC), kidney renal papillary cell carcinoma (KIRP), and kidney chromophobe carcinoma (KICH) being the most common subtypes. The Paired-box (PAX) gene family encodes transcription factors, which orchestrate multiple processes in cell lineage determination during embryonic development and organogenesis. Several PAX genes have been shown to be expressed in RCC following its onset and progression. Here, we performed real-time quantitative polymerase chain reaction (RT-qPCR) analysis on a series of human RCC cell lines, revealing significant co-expression of PAX2, PAX6, and PAX8. Knockdown of PAX2 or PAX8 mRNA expression using RNA interference (RNAi) in the A498 RCC cell line resulted in inhibition of cell proliferation, which aligns with our previous research, although no reduction in cell proliferation was observed using a PAX2 small interfering RNA (siRNA). We downloaded publicly available RNA-sequencing data and clinical histories of RCC patients from The Cancer Genome Atlas (TCGA) database. Based on the expression levels of PAX2, PAX6, and PAX8, RCC patients were categorized into two PAX expression subtypes, PAXClusterA and PAXClusterB, exhibiting significant differences in clinical characteristics. We found that the PAXClusterA expression subgroup was associated with favorable clinical outcomes and better overall survival. These findings provide novel insights into the association between PAX gene expression levels and clinical outcomes in RCC patients, potentially contributing to improved treatment strategies for RCC.

Utility of a PAX2, PTEN, and ß-catenin Panel in the Diagnosis of Atypical Hyperplasia/Endometrioid Intraepithelial Neoplasia in Endometrial Polyps.

The diagnosis of atypical hyperplasia/endometrioid intraepithelial neoplasm (AH/EIN) within endometrial polyps (EMPs) often poses a diagnostic conundrum. Our previous studies demonstrated that a panel of immunohistochemical (IHC) markers consisting of PAX2, PTEN, and ß-catenin can be effectively utilized for the identification of AH/EIN. A total of 105 AH/EIN within EMP were analyzed using the 3-marker panel. We also evaluated these cases for the presence of morules. Benign EMP (n=90) and AH/EIN unassociated with polyp (n=111) served as controls. Aberrant expression of PAX2, PTEN, or ß-catenin was observed in AH/EIN in EMP in 64.8%, 39.0%, and 61.9% of cases, respectively. At least 1 IHC marker was abnormal in 92.4% of cases. Overall, 60% of AH/EIN in EMP demonstrated abnormal results for≥2 IHC markers. The prevalence of PAX2 aberrancy was significantly lower in AH/EIN in EMP than in nonpolyp AH/EIN (64.8% vs. 81.1%, P =0.007), but higher than in benign EMP (64.8% vs. 14.4%, P <0.00001). The prevalence of ß-catenin aberrancy was significantly higher in AH/EIN in EMP than in nonpolyp AH/EIN (61.9% vs. 47.7%, P =0.037). All control benign EMP demonstrated normal expression of PTEN and ß-catenin. Morules were present in 38.1% of AH/EIN in EMP versus 24.3% in nonpolyp AH/EIN, and absent in benign EMP. A strong positive association was found between ß-catenin and morules (Φ=0.64). Overall, 90% cases of atypical polypoid adenomyoma (n=6) and mucinous papillary proliferation (n=4) showed IHC marker aberrancy. In conclusion, the 3-marker IHC panel (PAX2, PTEN, and ß-catenin) is (1) a useful tool in the diagnosis of AH/EIN in EMP; (2) PAX2 loss should be interpreted with caution and in combination with morphology and other markers.

Fgf, Hh, and pax2a differentially regulate expression of pax5 and pou3f3b in vestibular and auditory maculae in the zebrafish otic vesicle.

BACKGROUND: The vertebrate inner ear contains distinct sensory epithelia specialized for auditory or vestibular function. In zebrafish, the first sensory epithelia form at opposite ends of the otic vesicle and are functionally distinct: the anterior utricular macula is essential for vestibular function whereas the posterior saccular macula is critical for hearing. Mechanisms distinguishing these maculae are not clear. Here, we examined the effects of manipulating Fgf or Hh on expression of pax5 and pou3f3b, unique markers of utricular and saccular identity. We also examined the roles of pax2a and atoh1a/b, early regulators of sensory specification. RESULTS: fgf3 and fgf8a were uniquely required for pax5 and pou3f3b, respectively. Elevating Fgf or blocking Hh expanded expression of pax5 but repressed pou3f3b, while blocking Fgf had the opposite effect. Blocking sensory specification did not affect pax5 or pou3f3b, but both markers were lost in pax2a-/- mutants. Maintenance of pax2a expression requires Fgf, Hh and Pax2a itself. CONCLUSION: Specification of utricular identity requires high Fgf and is repressed by Hh, whereas saccular identity requires Hh plus low Fgf. pax2a acts downstream of Fgf and Hh to maintain both fates. Comparison with mouse suggests this may reflect a broadly conserved developmental mechanism.

Pax2a, Sp5a and Sp5l act downstream of Fgf and Wnt to coordinate sensory-neural patterning in the inner ear.

In zebrafish, sensory epithelia and neuroblasts of the inner ear form simultaneously in abutting medial and lateral domains, respectively, in the floor of the otic vesicle. Previous studies support regulatory roles for Fgf and Wnt, but how signaling is coordinated is poorly understood. We investigated this problem using pharmacological and transgenic methods to alter Fgf or Wnt signaling from early placodal stages to evaluate later changes in growth and patterning. Blocking Fgf at any stage reduces proliferation of otic tissue and terminates both sensory and neural specification. Wnt promotes proliferation in the otic vesicle but is not required for sensory or neural development. However, sustained overactivation of Wnt laterally expands sensory epithelia and blocks neurogenesis. pax2a, sp5a and sp5l are coregulated by Fgf and Wnt and show overlapping expression in the otic placode and vesicle. Gain- and loss-of-function studies show that these genes are together required for Wnt's suppression of neurogenesis, as well as some aspects of sensory development. Thus, pax2a, sp5a and sp5l are critical for mediating Fgf and Wnt signaling to promote spatially localized sensory and neural development.

Decreased Microglia in Pax2 Mutant Mice Leads to Impaired Learning and Memory.

Impaired learning and memory ability is one of the characteristics of a variety of neurological diseases, and its molecular mechanisms are complex and diverse and are regulated by a variety of factors. It is generally believed that synaptic plasticity plays an important role in the process of learning and memory. The protein encoded by the Pax2 gene is a transcription factor involved in neuron migration and cell fate determination during neural development. Mice knocked out of BDNF in the Pax2 lineage-derived interneuron precursor exhibited learning disabilities and severe cognitive impairment. In this study, Pax2 heterozygous gene (Pax2+/- mice) deletion mice were used as the research objects and behavioral tests were used to observe the effect of Pax2 gene deletion on learning and memory ability; morphological and molecular biological methods were used to observe the effect of Pax2 gene deletion on the neural structure. Single-cell transcriptome sequencing was used to observe the cell subtypes and differentially expressed genes (DEGs) and signaling pathways affected by Pax2 gene deletion and the possible molecular mechanisms. The results showed that Pax2+/- mice had impaired learning and memory ability, abnormal synaptic structure, and significantly reduced number of microglia clusters, and DEGs were associated with pro-inflammatory chemokines. Finally, we speculate that Pax2 gene deletion may lead to abnormal chemokines and chemokine receptors by affecting microglia.

The proliferative and the antifibrotic side of PAX2 in tubular repair.

Regenerative repair following injury to proximal tubular epithelial cells (PTECs) is essential to restore the kidney to normal function in acute kidney injury. Failure to accomplish this leads to chronic kidney disease. Expression of the paired-box transcription factor Pax2 in PTECs is required for their regenerative proliferation and repair. However, a loss-of-function study now shows that the absence of Pax2 not only impacts PTEC proliferation but also causes myofibroblast recruitment leading to excessive tubulointerstitial fibrosis.

PHF20L1 mediates PAX2 expression to promote angiogenesis and liver metastasis in colorectal cancer through regulating HIC1.

Colorectal cancer (CRC) is a common cancer with poor prognosis. The research was designed to explore the role of PHF20L1 in angiogenesis and liver metastasis in CRC and discuss its molecular mechanism. Expression levels of PHF20L1, HIC1 and PAX2 in CRC tissues collected from CRC patients were detected using qRT-PCR, WB and immunohistochemical staining. CRC cells were transfected with PHF20L1, HIC1 and PAX2 overexpression or knockdown vectors and the proliferation, apoptosis, EMT and angiogenesis of the cells were determined. WB was utilized to assess protein levels of PHF20L1, HIC1, PAX2 and angiogenesis factor (ANGPT2, FGF1, PDGFA and VEGFA). The role of PHF20L1 regulating tumor formation and liver metastasis in vivo was detected as well. PHF20L1 was observed to express at a high level of CRC tissues. PHF20L1 promoted CRC cell growth, EMT and angiogenesis, and inhibited cell apoptosis. Knockdown of PHF20L1 had opposite effects on CRC cells. PHF20L1 negatively regulated HIC1 expression to promote PAX2 expression, thus promoting CRC cell progression. The in vivo results showed that PHF20L1 contributed to tumor formation and liver metastasis. PHF20L1 increases PAX2 expression to promote angiogenesis in CRC by inhibiting HIC1, therefore facilitating CRC cell EMT and liver metastasis. Our finding may provide a novel insight for CRC pathogenesis.

Reliable Identification of Endometrial Precancers Through Combined Pax2, ß-Catenin, and Pten Immunohistochemistry.

The diagnosis of endometrial atypical hyperplasia/endometrioid intraepithelial neoplasia (AH/EIN) remains challenging and subjective in some cases, with variable histologic criteria and differences of opinion among gynecologic pathologists, potentially leading to under/overtreatment. There has been growing interest in the use of specific immunohistochemical markers as adjuncts in AH/EIN diagnosis. For example, the World Health Organization 2020 Classification specifies that loss of Pten, Pax2, or mismatch repair proteins are desirable diagnostic criteria. Other markers, most notably ß-catenin and Arid1a, are also aberrantly expressed in some AH/EIN. However, the performance of some markers individually-and more importantly as a group-has not been rigorously explored, raising questions as to which marker(s) or combination(s) is the most effective in practice. Formalin-fixed paraffin-embedded tissue sections from AH/EIN cases (n=111) were analyzed by immunohistochemistry for 6 markers: Pax2, Pten, Mlh1, ß-catenin, Arid1a, and p53. Aberrant expression was tabulated for each case and marker. An additional set of normal endometria (n=79) was also analyzed to define optimal diagnostic criteria for marker aberrance. The performance characteristics of each marker, the entire panel, and subsets thereof were quantitatively and statistically analyzed. In order of number of cases detected, the most frequently aberrant markers in AH/EIN were Pax2 (81.1% of cases), Pten (50.5%), ß-catenin (47.7%), Arid1a (7.2%), Mlh1 (4.5%), and p53 (2.7%). The majority of cases showed aberrant expression of ≥2 markers. All 6 markers together identified 92.8% of cases. Arid1a, Mlh1, and p53 were robust and readily scored markers, but all cases showing aberrant expression of these 3 markers were also detected by Pax2, Pten, or ß-catenin. A focused panel of only 3 markers (Pax2, Pten, and ß-catenin) showed optimal performance characteristics as a diagnostic adjunct in the histopathologic diagnosis of AH/EIN. Use of this panel is practicable and robust, with at least 1 of the 3 markers being aberrant in 92.8% of AH/EIN.

PAX2 promoter methylation and AIB1 overexpression promote tamoxifen resistance in breast carcinoma patients.

INTRODUCTION: Disease recurrence is an important obstacle in estrogen receptor positive (ER+) tamoxifen treated breast carcinoma patients. Tamoxifen resistance-related molecular mechanisms are not fully understood. Alteration in DNA methylation which contributes to transcriptional regulation of cancer-related genes plays a crucial role in tamoxifen response. In the present study, the contribution of promoter methylation and mRNA expression of PAX2 and AIB1 in the development of breast carcinoma and tamoxifen refractory was assessed. METHODS: Methylation specific-high resolution melting (MS-HRM) analysis and Real-time quantitative PCR (RT-qPCR) experiment were performed to analyze the promoter methylation and mRNA expression levels of PAX2 and AIB1 genes in 102 breast tumors and adjacent normal breast specimens. RESULTS: We indicated that PAX2 expression is decreased in breast tissues due to hypermethylation in its promoter region. Compared to the adjacent normal tissues, the tumors exhibited significantly lower relative mRNA levels of PAX2 and increased expression of AIB1. Aberrant promoter methylation of PAX2 and overexpression of AIB1 was observed in tamoxifen resistance patients compared to the sensitive ones. Cox regression analysis exhibited that the increased promoter methylation status of PAX2 and overexpression of AIB1 remained as unfavorable identifiers which influence patients' survival independently. CONCLUSIONS: Our results revealed that the aberration in PAX2 promoter methylation and AIB1 overexpression are associated with the tamoxifen response in breast carcinoma patients. Further research is needed to demonstrate the potential of using PAX2 and AIB1 expression and their methylation-mediated regulation as predictive or prognostic biomarkers or as a new target therapy for better disease management.

Dynamics of muscle growth and regeneration: Lessons from the teleost.

The processes of myogenesis during both development and regeneration share a number of similarities across both amniotes and teleosts. In amniotes, the process of muscle formation is considered largely biphasic, with developmental myogenesis occurring through hyperplastic fibre deposition and postnatal muscle growth driven through hypertrophy of existing fibres. In contrast, teleosts continue generating new muscle fibres during adult myogenesis through a process of eternal hyperplasia using a dedicated stem cell system termed the external cell layer. During developmental and regenerative myogenesis alike, muscle progenitors interact with their niche to receive cues guiding their transition into myoblasts and ultimately mature myofibres. During development, muscle precursors receive input from neighbouring embryological tissues; however, during repair, this role is fulfilled by other injury resident cell types, such as those of the innate immune response. Recent work has focused on the role of macrophages as a pro-regenerative cell type which provides input to muscle satellite cells during regenerative myogenesis. As zebrafish harbour a satellite cell system analogous to that of mammals, the processes of regeneration can be interrogated in vivo with the imaging intensive approaches afforded in the zebrafish system. This review discusses the strengths of zebrafish with a focus on both the similarities and differences to amniote myogenesis during both development and repair.

Pax2a, but not pax2b, influences cell survival and periocular mesenchyme localization to facilitate zebrafish optic fissure closure.

BACKGROUND: Pax2 is required for optic fissure development in many organisms, including humans and zebrafish. Zebrafish loss-of-function mutations in pax2a display coloboma, yet the etiology of the morphogenetic defects is unclear. Further, pax2 is duplicated in zebrafish, and a role for pax2b in optic fissure development has not been examined. RESULTS: Using a combination of imaging and molecular genetics, we interrogated a potential role for pax2b and examined how loss of pax2 affects optic fissure development. Although optic fissure formation appears normal in pax2 mutants, an endothelial-specific subset of periocular mesenchyme (POM) fails to initially localize within the optic fissure, yet both neural crest and endothelial-derived POM ectopically accumulate at later stages in pax2a and pax2a; pax2b mutants. Apoptosis is not up-regulated within the optic fissure in pax2 mutants, yet cell death is increased in tissues outside of the optic fissure, and when apoptosis is inhibited, coloboma is partially rescued. In contrast to pax2a, loss of pax2b does not appear to affect optic fissure morphogenesis. CONCLUSIONS: Our results suggest that pax2a, but not pax2b, supports cell survival outside of the optic fissure and POM abundance within it to facilitate optic fissure closure.

Incidence of malignant transformation in the oviductal fimbria in laying hens, a preclinical model of spontaneous ovarian cancer.

Ovarian high grade serous carcinoma (HGSC) is a lethal form of ovarian cancer (OVCA). In most cases it is detected at late stages as the symptoms are non-specific during early stages. Emerging information suggests that the oviductal fimbria is a site of origin of ovarian HGSC. Currently available tests cannot detect ovarian HGSC at early stage. The lack of a preclinical model with oviductal fimbria that develops spontaneous ovarian HGSC is a significant barrier to developing an early detection test for this disease. The goal of this study was to examine if the oviductal fimbria in hens is a site of origin of HGSC and whether it expresses several putative markers expressed in ovarian HGSC in patients. A total of 135 laying hens (4 years old) were selected from a flock using transvaginal ultrasound (TVUS) imaging, followed by euthanasia and gross examination for the presence of solid masses and ascites. Histological types of carcinomas were determined by hematoxylin and eosin staining. Expression of WT-1, mutant p53, CA-125, PAX2 and Ki67 in normal or malignant fimbriae or ovaries were examined using immunohistochemistry, immunoblotting and gene expression assays. This study detected tumors in oviductal fimbriae in hens and routine staining revealed ovarian HGSC-like microscopic features in these tumors. These tumors showed similarities to ovarian HGSC in patients in expressing several markers. Compared with normal fimbriae, intensities of expression of WT-1, mutant p53, CA-125, and Ki67 were significantly (P<0.05) higher in fimbrial tumors. In contrast, expression of PAX2 decreased gradually as the tumor progressed to late stages. The patterns of expression of these markers were similar to those in ovarian HGSC patients. Thus, tumors of the oviductal fimbria in hens may offer a preclinical model to study different aspects of spontaneous ovarian HGSC in women including its early detection.

Impact of WNT Pathway Blockade on the Biological Characteristics of Renal Tubular Epithelial Cells Stably Transfected with PAX2.

OBJECTIVE: The WNT pathway might be the primary pathway for the regulation of renal development by PAX2. In this study, we aimed to observe the migration and invasion abilities of human tubular epithelial cells stably transfected with PAX2 upon the WNT pathway blockade and to investigate whether the WNT pathway is involved in the regulation of cellular biological activity by PAX2. METHODS: 1. Control cells-PAX2 and control cells+PAX2 groups were formed for monitoring the expression of PAX2 and ß-catenin mRNA using RT-qPCR. 2. The PAX2-expressing cells were treated with WIF-1 (5 µg/ml), WIF-1 (10 µg/ml), or WIF-1 (15 µg/ml), and the effect was analyzed by Western blotting analysis after the WNT pathway blockade. 3. The migration and invasion abilities of the PAX2-expressing cells were evaluated using cell-scratch and transwell assays after blocking the WNT pathway. RESULTS: 1. RT-qPCR: The expression of PAX2 and ß-catenin increased significantly in the PAX2-expressing cells (P<0.05). 2. Upon treatment with WIF-1, the expression of ß-catenin in the PAX2 cells+WIF-1 5 µg/ml group, PAX2 cells+WIF-1 10µg/ml group, and PAX2 cells+WIF-1 15 µg/ml group decreased significantly compared to in the PAX2 cells-WIF-1 group (P<0.05), especially the WIF-1 (15 µg/ml) group (P<0.05). 3. The cell migration rate in the PAX2 cells + WIF-1 (15 µg/ml) group at 18 h was significantly lower than that in the PAX2 cells-WIF-1 group (P<0.05). 3. Transwell assay: the invasion ability in the PAX2 cells+WIF-1 (15 µg/ml) group was lower than that in the PAX2 cells-WIF-1 group (P<0.05). CONCLUSION: WNT pathway blockade can weaken the migration and invasion abilities of PAX2-expressing cells. Moreover, the WNT pathway was observed to be associated with the regulation of cellular biological activity by PAX2.

Silencing PTEN in the fallopian tube promotes enrichment of cancer stem cell-like function through loss of PAX2.

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy that is primarily detected at the metastatic stage. Most HGSOC originates from the fallopian tube epithelium (FTE) and metastasizes to the ovary before invading the peritoneum; therefore, it is crucial to study disease initiation and progression using FTE-derived models. We previously demonstrated that loss of PTEN from the FTE leads to ovarian cancer. In the present study, loss of PTEN in FTE led to the enrichment of cancer stem cell markers such as LGR5, WNT4, ALDH1, CD44. Interestingly, loss of the transcription factor PAX2, which is a common and early alteration in HGSOC, played a pivotal role in the expression of cancer stem-like cells (CSC) markers and cell function. In addition, loss of PTEN led to the generation of two distinct subpopulations of cells with different CSC marker expression, tumorigenicity, and chemoresistance profiles. Taken together, these data suggest that loss of PTEN induces reprogramming of the FTE cells into a more stem-like phenotype due to loss of PAX2 and provides a model to study early events during the FTE-driven ovarian cancer tumor formation.

Spinal Excitatory Dynorphinergic Interneurons Contribute to Burn Injury-Induced Nociception Mediated by Phosphorylated Histone 3 at Serine 10 in Rodents.

The phosphorylation of serine 10 in histone 3 (p-S10H3) has recently been demonstrated to participate in spinal nociceptive processing. However, superficial dorsal horn (SDH) neurons involved in p-S10H3-mediated nociception have not been fully characterized. In the present work, we combined immunohistochemistry, in situ hybridization with the retrograde labeling of projection neurons to reveal the subset of dorsal horn neurons presenting an elevated level of p-S10H3 in response to noxious heat (60 °C), causing burn injury. Projection neurons only represented a small percentage (5%) of p-S10H3-positive cells, while the greater part of them belonged to excitatory SDH interneurons. The combined immunolabeling of p-S10H3 with markers of already established interneuronal classes of the SDH revealed that the largest subset of neurons with burn injury-induced p-S10H3 expression was dynorphin immunopositive in mice. Furthermore, the majority of p-S10H3-expressing dynorphinergic neurons proved to be excitatory, as they lacked Pax-2 and showed Lmx1b-immunopositivity. Thus, we showed that neurochemically heterogeneous SDH neurons exhibit the upregulation of p-S10H3 shortly after noxious heat-induced burn injury and consequential tissue damage, and that a dedicated subset of excitatory dynorphinergic neurons is likely a key player in the development of central sensitization via the p-S10H3 mediated pathway.

Epigenetic regulation of arginine vasopressin receptor 2 expression by PAX2 and Pax transcription interacting protein.

Renal arginine vasopressin receptor 2 (AVPR2) plays a crucial role in osmoregulation. Engagement of ligand with AVPR2 results in aquaporin 2 movement to the apical membrane and water reabsorption from the urinary filtrate. Despite this essential role, little is known about transcriptional regulation of Avpr2. Here, we identify novel roles for PAX2, a transcription factor crucial for kidney development, and its adaptor protein, Pax transcription interacting protein (PTIP), for epigenetic regulation of Avpr2 and thus body water balance. Chromatin immunoprecipitation (ChIP) from murine inner medulla cells (IMCD-3) identified the minimal DNA-binding region of PAX2 on the Avpr2 promoter. Regulation of Avpr2 by PAX2 was confirmed using a heterologous DNA expression system. PAX2 recruits the adaptor protein PTIP and its associated histone methyltransferase (HMT) complex to Avpr2 promoter, imposing epigenetic marks on this region and throughout the coding sequence that modulate Avpr2 gene transcription. Reduction of PAX2 or PTIP protein levels by siRNA prevented histone lysine methylation and expression of Avpr2. ChIP using mouse or human kidneys determined that PAX2 is highly enriched in the AVPR2 promoter alongside PTIP and HMT proteins, leading to high levels of histone H3 lysine trimethylation within the promoter and throughout the gene. In conclusion, PAX2 provides locus specificity for PTIP, allowing the HMT complex to impart epigenetic changes at the Avpr2 locus and regulate Avpr2 transcription. These finding have major implications for understanding regulation of body water balance.NEW & NOTEWORTHY The transcription factor PAX2 plays an indispensable role in kidney development. In the adult kidney, we identified the first described protein this protein regulates. PAX2 and its interacting partner Pax transcription interacting protein recruit a histone methyltransferase complex to the promoter and epigentically regulate the expression of arginine vasopressin receptor 2, a protein that plays a crucial role in osmoregulation in the distal tubule.