Depletion of ALMS1 affects TGF-β signalling pathway and downstream processes such as cell migration and adhesion capacity

Abstract

Background: ALMS1 is a ubiquitous gene associated with Alström syndrome (ALMS). The main symptoms of ALMS affect multiple organs and tissues, generating at last, multi-organic fibrosis in the lungs, kidneys and liver. TGF-β is one of the main pathways implicated in fibrosis, controlling the cell cycle, apoptosis, cell migration, and epithelial-mesenchymal transition (EMT). Nevertheless, the role of ALMS1 gene in fibrosis generation and other implicated processes such as cell migration or epithelial-mesenchymal transition via the TGF-β pathway has not been elucidated yet. Methods: Initially, we evaluated how depletion of ALMS1 affects different processes like apoptosis, cell cycle and mitochondrial activity. Then, we performed proteomic profiling with TGF-β stimuli in HeLa ALMS1 -/- cells and validated the results by examining different EMT biomarkers using qPCR. The expression of these EMT biomarkers was validated in hTERT-BJ-5ta. Finally, we also evaluated the SMAD3 phosphorylation in BJ-5ta model and its cell migration capacity. Results: Depletion of ALMS1 generated apoptosis resistance to thapsigargin (THAP) and C2-Ceramide (C2-C), and G2/M cell cycle arrest in HeLa cells. For mitochondrial activity, results did not show significant differences between ALMS1 +/+ and ALMS1 -/-. Proteomic results showed inhibition of downstream pathways regulated by TGF-β. The protein-coding genes (PCG) were associated with processes like focal adhesion or cell-substrate adherens junction. EMT biomarkers VIM, DSP, and SNAI1 showed an opposite pattern to what would be expected when activating the EMT. Finally, inhibition of SNAI1 was consistent in BJ-5ta model, where a reduced activation of SMAD3 and a decrease in migratory capacity were also observed. Conclusion: ALMS1 has a role in controlling the cell cycle and the apoptosis processes. Moreover, the depletion of ALMS1 inhibits the signal transduction through the TGF-β/SMAD3/SNAI1, which could be affecting the cell migration capacity and EMT.

Loss of the centrosomal protein ALMS1 alters lipid metabolism and the regulation of extracellular matrix-related processes

“…The inhibition of p53 would be caused by this over-activation through MDM2, affecting the PTEN-p53-AKT-MDM2 loop (Gottlieb et al, 2002;Abraham and O'Neill, 2014). This hypothesis would be supported by the resistance to apoptosis in ALMS1-depleted cells (Zulato et al, 2011;Bea-Mascato et al, 2022).…”

“…For this reason, it is timely to determine the role of different ciliary and basal body genes, such as ALMS1, in their regulation. Thus, we further explored the previously described association between ALMS1 deficiency and alterations in TGF-β-mediated signal transduction (Álvarez-Satta et al, 2021;Bea-Mascato et al, 2022). We performed gene expression profiling by RNA-seq and LFQ-proteomics in an ALMS1KO hTERT-BJ-5ta cell line stimulated with TGF-β1.…”

“…The copyright holder for this this version posted November 2, 2022. ; https://doi.org/10.1101/2022.11.02.514847 doi: bioRxiv preprint foreskin fibroblasts (hFF) after TGF-β activation (Mönnich et al, 2018). However, these alterations were not observed in ALMS1 knockout models in HeLa or hTERT-BJ-5ta (Bea-Mascato et al, 2022). In these models the only alteration detected in the canonical pathway was a slight decrease in SMAD3 phosphorylation/activation in the hTERT-BJ-5ta model (Bea-Mascato et al, 2022).…”

Phosphoproteomic profiling highlights CDC42 and CDK2 as key players in the regulation of the TGF-β pathway inALMS1andBBS1knockout models

“…The hTERT-BJ-5ta cell line, provided by the American Type Culture Collection (ATCC), was maintained in a medium composed of 4:1 of Dulbecco’s minimal essential medium (DMEM, Gibco, Invitrogen, NY, USA) and Medium 199, Earle’s Salts (Gibco, Invitrogen, NY, USA), supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, NY, USA) and 2% penicillin/streptomycin (P/S) (Gibco, Invitrogen, NY, USA). In this study, a knockout model for the BBS1 gene was generated (Figure S1 A, B) using 4 different sgRNAs (Supplementary Table S1-2) and a knockout (KO) model for the ALMS1 gene previously generated in Bea-Mascato et al (Bea-Mascato et al, 2022b) was used. The methodology used in the generation of the BBS1 model was the same as described in Bea-Mascato et al (Bea-Mascato et al, 2022b).…”

“…It is related to the control of several processes such as mitosis, apoptosis, cell migration or autophagy through cross-signalling with other pathways such as MAPK, PI3K/AKT or SMADs (Massagué, 2012; Hamidi et al, 2017; Luo, 2017; Zhang et al, 2017; Gasior et al, 2019). In previous studies in our laboratory, we have determined that ALMS1 depletion leads to altered signal transduction of the TGF-β pathway and other related pathways such as AKT or p53 (Álvarez-Satta et al, 2021; Bea-Mascato et al, 2022b, 2022a), altering processes such as cell migration or EMT. In this study, we apply an original approach based on phospho-proteomics to delve into the regulation of the TGF-β pathway in ALMS and to determine for the first time whether there are alterations in this pathway in BBS.…”