Biology subjects

O'Donnell, B. L.

Publications and source records attributed to O'Donnell, B. L..

8 recordsLinked to original sources

bioRxiv2026

Pannexin 1 Ablation Disrupts Cell Cycle Progression and Compromises the Structure of Melanoma Tumours

Pannexin 1 (PANX1) is a channel-forming glycoprotein that has been implicated in regulating melanoma cell properties such as growth, migration, and invasion. Here we report that the CRISPR-Cas9 mediated ablation of PANX1 or channel inhibition with probenecid shifted A375-P cell cycle distribution towards G2/M, while channel inhibition with spironolactone stalled cell cycle progression at the G1/S checkpoint regardless of PANX1 expression. Bulk RNA sequencing revealed cell cycle related gene expression was downregulated with PANX1 targeting strategies and this was exacerbated when PANX1 deficient cells were treated with spironolactone. When grown as spheroids, we found the deletion and inhibition of PANX1 disrupted spheroid compaction, with spheroids displaying poorly defined borders and reduced circularity. The deletion of PANX1 increased spheroid fragmentation, and this phenotype was intensified when spheroids devoid of PANX1 were treated with spironolactone, highlighting a potential synthetic lethal effect. Using liquid chromatography mass spectrometry, we sought to determine whether PANX1 had binding partners that may act to stabilize spheroid integrity and found that PANX1 co-immunoprecipitated various cell adhesion molecules such as {beta}-actin and F-actin-capping protein subunit -1, suggesting PANX1 and its interacting partners may stabilize cell-cell contacts. When control and PANX1 ablated cells were grown in chick chorioallantoic membrane xenografts to assess their tumour growth characteristics, we discovered that the deletion of PANX1 reduced tumour surface area and viability. Our findings indicate that PANX1 regulates cell cycle progression and the structural architecture of A375-P melanoma tumours, highlighting the therapeutic utility of targeting PANX1 in melanoma.

cancer biology↗

bioRxiv2026

Pannexin 1 phosphorylation sites differentially modulate channel activity and physiological outcomes

Within the vasculature, pannexin 1 (PANX1) channels in smooth muscle cells (SMCs) regulate -adrenergic constriction and blood pressure. PANX1 channel activity is regulated by phosphorylation at Y198, S205 and Y308 residues, but the physiological significance of these modifications is unknown. Here, we utilize newly developed PANX1 Y198F, S205A and Y308F phospho-dead mutant mice to test physiological changes related to hemodynamics. Radiotelemetry-measured blood pressure was decreased in Y198F, increased in Y308F, but unchanged in S205A mice at baseline. Clonidine-sensitive sympathetic-driven hypertension was observed in all mouse lines except Y198F. Pressure myography of third-order mesenteric arteries revealed -adrenergic contractile responses were decreased in Y198F, slightly enhanced in Y308F, but unchanged in S205A, with responses in Y198F vessels mimicking controls treated with PANX1 inhibitors. To understand signaling changes driving these phenotypes, we performed mesenteric artery bulk RNA sequencing, but found a minimal number of differentially expressed genes between phospho-dead mutants and controls. Similarly, co-immunoprecipitation-mass spectrometry of wildtype or phospho-dead mutant-expressing vascular SMCs revealed few interacting proteins distinct to each PANX1 variant. However, PANX1 channel activity assessments in HEK293T cells expressing the 1D-adrenergic receptor as well as each phospho-dead mutant PANX1 showed that phenylephrine-induced ATP release from Y198F channels was significantly decreased compared to wildtype, but current was unaffected. Conversely, basal and phenylephrine-induced S205A and Y308F currents were reduced, but ATP release resembled controls. Taken together, these findings indicate that distinct PANX1 phosphorylation determines PANX1 metabolite release versus current conducting properties and in turn, regulates physiological outcomes in the vasculature. One Sentence SummaryPANX1 Y198 phosphorylation-mediated ATP release is a major driver of -adrenergic vasoconstriction in vascular smooth muscle cells.

physiology↗

bioRxiv2025

Arterial iron regulates vasodilation during anemia via endothelial holo-alpha globin

Iron deficiency is a highly prevalent nutrient deficiency and the most common cause of anemia. Although iron deficiency exacerbates cardiovascular disease, the direct impact of iron deficiency on the vasculature remains unstudied. We assessed iron levels across the vascular endothelium and found resistance artery endothelial cells to have the lowest iron stores suggesting they may be especially impacted by iron deficiency. Anemia has previously been shown to increase arterial NO signaling in patients, and we have previously shown endothelial -globin (Hb) scavenges nitric oxide (NO) in the resistance artery endothelium. We hypothesize iron regulates vascular function through downregulation of endothelial Hb. To test this, we used a novel model of iron deficiency anemia (IDA). In female mice, IDA increased NO signaling which was rescued to control levels by repletion of vascular iron with ferric dextran. Despite being similarly anemic and having a similar reduction in Hb protein, there were no changes in NO signaling across groups in male mice. We further measured whether Hb was in its heme-bound (holo-Hb) or heme-free (apo-Hb) state and found males did not fully lose holo-Hb. Using endothelial specific Hb knockout mice, we show loss of endothelial Hb is necessary for increased NO signaling in IDA and for the rescue of NO signaling by ferric dextran in female mice. Altogether the data presented here demonstrate iron modulates endothelial NO signaling through the regulation of Hb.

physiology↗

bioRxiv2024

Novel Pannexin 1 isoform is increased in cancer

Alternative translation initiation (ATI) is a process of increasing protein diversity from one transcript, allowing cells to rapidly respond to signals, which is particularly important in cancer cells. Here, we report potential internal translation start sites exist in PANX1 which have implications in trafficking and channel function. Using mouse (mPANX1) constructs for each internal methionine, we saw that these PANX1 isoforms were N-glycosylated, could traffic to the cell surface and mPANX1-M37 formed functional channels activated by C-terminus cleavage or 1-adrenoceptor stimulation. We also identified a ~25 kDa isoform of mPANX1 (mPANX1-25K) endogenously expressed in mouse melanoma cell lines that could be confirmed with a cognate peptide. mPANX1-25K lacks the mPANX1 N-terminus and most likely corresponds to the M210 internal translation start site since we could not identify any alternative transcripts that would produce this ATI product. When we expressed the human equivalent of M210 in Hs578T PANX1 KO cells with and without wildtype human PANX1, we determined M211 exhibits a predominantly intracellular localization, is N-glycosylated and can interact with full-length human PANX1. Collectively, these findings indicate species specific differences in the abundance of PANX1 ATI isoforms which could act independently or in conjunction with the canonical full-length protein in melanoma.

cell biology↗

bioRxiv2024

Pannexin 1 crosstalk with the Hippo pathway in malignant melanoma

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, YAP, TAZ, and Hippo scaffold, IQGAP1, in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels. Notably, our investigations uncovered a previously unrecognized interaction between endogenous PANX1 and the Hippo scaffold protein IQGAP1 in melanoma cells. Moreover, our findings revealed that IQGAP1 exhibits differential expression in melanoma cells and plays a regulatory role in cellular morphology. Functional studies involving PANX1 knockdown provided compelling evidence that PANX1 modulates YAP protein levels and its co-transcriptional activity in both melanoma and breast carcinoma cells. Importantly, our study showcases the potential therapeutic relevance of targeting PANX1, as pharmacological inhibition of PANX1 using selective FDA-approved inhibitors or PANX1 knockdown reduced YAP abundance in melanoma cells. Furthermore, our Clariom S analysis unveiled key genes implicated in cell proliferation, such as neuroglin1 (NRG1), {beta}-galactoside binding protein, galectin-3 (LGALS3), that are affected in PANX1- deficient cells. In summary, our investigation delves into the intricate interplay between PANX1 and YAP in the context of invasive melanoma, offering valuable insights into potential therapeutic strategies for effective treatment.

cell biology↗

bioRxiv2024

Pannexin 1 and Pannexin 3 differentially regulate the tumorigenic properties of cutaneous squamous cell carcinoma

Pannexin (PANX) channels are present in skin and facilitate the movement of signalling molecules during cellular communication. PANX1 and PANX3 function in skin homeostasis and keratinocyte differentiation but were previously reduced in a small cohort of human cutaneous squamous cell carcinoma (cSCC) tumours compared to normal epidermis. In our study, we used SCC-13 cells, limited publicly available RNA-seq data and a larger cohort of cSCC patient-matched samples to analyze PANX1 and PANX3 expression and determine the association between their dysregulation and the malignant properties of cSCC. In a bioinformatics analysis, PANX1 transcripts were increased in cSCC and head and neck SCC tumours compared to normal tissues, but PANX3 mRNA showed no differences. However, in our own cohort, PANX3 transcripts were decreased in cSCC compared to patient-matched aged skin, whereas PANX1 was upregulated in cSCC. PANX1 localized to all regions within the cSCC tumour microenvironment and increased levels were associated with larger tumour dimensions. To investigate PANX1 function in SCC-13 cells, we deleted PANX1 via CRISPR/Cas9 and treated with PANX1 inhibitors which markedly reduced cell growth and migration. To assess PANX3 function in cutaneous carcinogenesis, we employed the DMBA/TPA model using our global Panx3 knockout (KO) mice, where 60% of wildtype and 100% of KO mice formed pre-cancerous papillomas. Average papilloma volumes at endpoint were significantly increased in KO mice and showed moderate evidence of increases in KO mice over time. Collectively, these findings suggest PANX1 and PANX3 dysregulation may have potential tumour promoting and suppressive effects for keratinocyte transformation, respectively. Key points summaryO_LIPannexin 1 and pannexin 3 are channel-forming proteins which are critical in the normal maintenance and function of keratinocytes in the skin but may become altered in cutaneous squamous cell carcinoma (cSCC) tumours. C_LIO_LIIn this study, we used a combination of culture models, mouse models and patient-derived tissues. We found pannexin 1 levels are increased in cSCC tumours and present in all tumour regions, functioning to promote cSCC cell growth and migration. C_LIO_LIConversely, pannexin 3 levels are decreased in cSCC tumours and this protein reduces the incidence and growth of pre-cancerous lesions. C_LIO_LITaken together, our data indicates that in cSCC these pannexin family members seem to have opposite effects, where pannexin 1 is pro-tumorigenic and pannexin 3 is anti-tumorigenic. C_LIO_LIThese results help us to better understand the mechanisms of malignant transformation of keratinocytes and offer a new potential therapeutic target for the treatment of advanced cSCC. C_LI

cancer biology↗

bioRxiv2023

Aged male and female Panx3 KO mice develop severe osteoarthritis independent of forced mechanical use.

Osteoarthritis (OA) is a multi-factorial disease associated with aging. As the molecular mechanisms underpinning the pathogenesis of this disease are unclear, there are no disease-modifying drugs to combat OA. Pannexin 3 (PANX3) has been shown to promote cartilage loss during posttraumatic OA. In contrast, the ablation of Panx3 in male mice results in spontaneous full-thickness cartilage lesions at 24 months of age. While protected from traumatic intervertebral disc (IVD) degeneration, Panx3 knockout (KO) mice show signs of IVD disease with altered disc mechanics. Whether the deleterious effects of ablating Panx3 in aging is the result from accumulated mechanical damage is unknown. We used male and female wildtype (WT) and global Panx3 KO C57Bl6 mice aged to 18 months of age. Mice were then randomized to sedentary (SED) or forced treadmill running (FEX) for 6 weeks. Knee joint tissues including patellar tendon, quadriceps and distal patellar enthesis, and synovium were analyzed histologically and through micro-CT, along with lumbar spine IVDs. Half of male and female sedentary Panx3 KO mice developed full-thickness cartilage lesions, severe synovitis, and ectopic fibrocartilage deposition and calcification of the knee joints in comparison to all other conditions. Panx3 KO mice with severe OA show signs of quadriceps and patellar enthesitis, characterized by bone and marrow formation. Forced treadmill running did not seem to exacerbate these phenotypes in male or female Panx3 KO mice; however, it may have contributed to the development of lateral compartment OA. The IVDs of aged Panx3 KO mice displayed no apparent differences to control mice, and forced treadmill running had no further effects in either genotype. We conclude that aged Panx3 KO mice show features of late-stage primary OA including full-thickness cartilage erosion, severe synovitis, and enthesitis. These data suggest that the deletion of Panx3 is deleterious to synovial joint health in aging. LAY SUMMARYOsteoarthritis is a common joint disease, and its incidence can increase with aging. As such, no drugs are available to treat its progression. Recently, we discovered that cellular channels composed of Pannexin 3 (PANX3) proteins may contribute to the health of joint tissues. In this study, we propose a new model for age-related primary OA, using histology, microCT, and pathology assessments under sedentary and forced exercise conditions. We found that aged mice lacking the channel protein PANX3 developed late-stage OA, including severe cartilage loss, joint inflammation, and tendon attachment damage. Half of the sedentary PANX3-deficient mice showed these changes, along with abnormal bone growth. Treadmill running did not make the condition worse but may have caused additional joint damage in some areas. The spine, however, remained unaffected. These findings suggest that deleting Panx3 harms joint health with aging. Importantly, this mouse model mimics human primary OA, even without added factors like diet changes or forced exercise.

physiology↗

bioRxiv2022

Pannexin 3 channels regulate architecture, adhesion, barrier function and inflammation in the skin

The channel-forming glycoprotein Pannexin 3 (PANX3) functions in cutaneous wound healing and keratinocyte differentiation, but its role in skin homeostasis through aging is not yet understood. We found that PANX3 is absent in newborn skin but becomes upregulated with age. We characterized the skin of global Panx3 knockout mice (KO) and found that KO dorsal skin showed sex-differences at different ages, but generally had reduced dermal and hypodermal areas compared to aged-matched controls. Transcriptomic analysis of KO epidermis revealed reduced E-cadherin stabilization and Wnt signaling compared to WT, consistent with the inability of primary KO keratinocytes to adhere in culture, and diminished epidermal barrier function in KO mice. We also observed increased inflammatory signaling in KO epidermis and higher incidence of dermatitis in aged KO mice compared to wildtype controls. These findings suggest that during skin aging, PANX3 is critical in the maintenance of dorsal skin architecture, keratinocyte cell-cell and cell-matrix adhesion and inflammatory skin responses.

cell biology↗