Biology subjects

Solarat, C.

Publications and source records attributed to Solarat, C..

2 recordsLinked to original sources

bioRxiv2024

Depletion of BBSome Subunits Alters Receptor Endocytosis and Promotes EMT via TGF-β Signaling

Background: Ciliopathies are genetic disorders caused by defects in the structure or function of the cilia and their related structures. Bardet-Biedl syndrome (BBS) is a complex ciliopathy with varied symptoms, probably due to altered membrane receptor signalling pathways. Methods: We investigated the role of BBS1 and BBS4 gene deficiencies in retinal epithelial cells using surface membrane proteomics, co-immunoprecipitation, AlphaFold structural modelling, endocytic trafficking colocalization assays, autophagy flux analysis, and epithelial-to-mesenchymal transition (EMT) functional assays including migration and wound healing. Results: Deficiencies in these BBSome components led to shorter cilia and disrupted receptor endocytosis, with accumulation of TGF-{beta}-pathway and EMT-associated proteins on the plasma membrane of BBS1 KO cells. Co-immunoprecipitation and AlphaFold structural modelling indicated that neither BBS1 nor BBS4 forms stable physical complexes with core endocytic components (EEA1, RAB11, RAB7, LAMP2), suggesting an indirect mechanism. BBS1 KO cells showed sustained TGFBR1 recycling, low basal autophagic flux that increases upon TGF-{beta} stimulation, and the most pronounced mesenchymal phenotype. BBS4 KO cells showed reduced receptor recycling, elevated basal autophagic flux that declines upon stimulation, and the strongest canonical TGF-{beta}/SMAD activation with reduced non-canonical ERK1/2 signalling. Conclusions: Increased EMT markers and enhanced migration in BBS1 KO cells highlight the role of BBSome-dependent receptor trafficking as a potential therapeutic target for retinal degeneration in BBS. The divergent autophagic and signalling phenotypes between BBS1 and BBS4 KO reveal subunit-specific contributions to TGF-{beta} pathway dysregulation.

molecular biology↗

bioRxiv2021

Common variation in EDN1 regulatory regions highlights the role of PPARγ as a key regulator of Endothelin in vitro

Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the commonest targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of PAH patients, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors predicted for these regions (PPAR{gamma}, KLF4, and VDR) to assess Endothelin-1 expression in cell culture and validate the binding sites. First, we detected a SNP in the 5UTR (rs397751713) and another in the 3regulatory region (rs2859338) that increased luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPAR{gamma} could be binding in the rs397751713 and VDR in rs2859338. By using siRNAs and luciferase, we determined that PPAR{gamma} binds differentially in rs397751713. PPAR{gamma} and VDR Knock-Down, increased EDN1 mRNA levels and Endothelin-1 production in PAECs, while PPAR{gamma} and KLF4 Knock-Down increased the Endothelin-1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter Endothelin-1 levels. We were able to validate that PPAR{gamma} binds in rs397751713 and is key to regulate Endothelin-1. Also, KLF4 and VDR regulate Endothelin-1 production in a cell-dependent manner, but for VDR this interaction does not happen by binding directly to the regions we studied.

cell biology↗