A role for thyroid receptor isoforms in mitochondrial bioenergetic capacity of human adipose-derived stem cell

Abstract

Thyroid hormone receptors (TRs) α and β are homologous ligand-dependent transcription factors. We recently found that TRα is a predominant form in human adipose derived stem cells (hADSC). Further, we demonstrated that major TRα variants, TRα1 and TRα2 display mainly cytoplasmic distribution and colocalize with mitochondria. In this study, we investigated possible role for TRα receptor isoforms in mitochondrial bioenergetic capacity of hADSC. hADSC were purchased from Invitrogen, and all donors were non-diabetic females. Cells were plated in MesenPRO RS medium and grown to 50% confluency. To knock down (KD) particular TRα isoforms we used custom SMART pool ON-TARGET plus TRα1 or TRα2 siRNA, at 50 nM final concentration for 4 days. ICC was performed on cells cultured in 4-well chamber slides. Cells were fixed with 4% PFA for 20 minutes, rinsed three times with PBS and then permeabilized in 0.1% Triton X-100 dissolved in PBS for 10 minutes. Subsequently, cells were blocked for 30 minutes using 10% goat serum in PBS. Cells were incubated with mixture of fluorophore-conjugated mouse monoclonal antibodies against ATP synthase subunit beta (ATPB, Alexa Fluor 488) and ATPase inhibitory factor 1 (IF1, Alexa Fluor 647), and analyzed using a Leica TCS SP5 II confocal microscope. The colocalization rate and relative fluorescence intensities of ATPB/IF1 were determined at several regions of interest using LAS AF software. Our data revealed significant increase in ATPB and IF1 immunopositivity in both TRα1 KD and TRα2 KD cells compared to control. Elevated levels of ATPB and IF1 after TRα1 or TRα2 knockdown could be a result of compensatory mechanism due to suppression of one the TRα isoforms. This study implicates both TRα isoforms as regulators of hADSCs mitochondrial bioenergetic capacity, and further research is needed to elucidate that role.