Abstract

The tumor suppressor phosphatase and tensin homolog (PTEN) negatively regulates the insulin signaling pathway. Germline PTEN pathogenic variants cause PTEN hamartoma tumor syndrome (PHTS), associated with lipoma development in children. Adipose progenitor cells (APCs) lose their capacity to differentiate into adipocytes during continuous culture, whereas APCs from lipomas of patients with PHTS retain their adipogenic potential over a prolonged period. It remains unclear which mechanisms trigger this aberrant adipose tissue growth. To investigate the role of PTEN in adipose tissue development, we performed functional assays and RNA-Seq of control and PTEN knockdown APCs. Reduction of PTEN levels using siRNA or CRISPR led to enhanced proliferation and differentiation of APCs. Forkhead box protein O1 (FOXO1) transcriptional activity is known to be regulated by insulin signaling, and FOXO1 was downregulated at the mRNA level while its inactivation through phosphorylation increased. FOXO1 phosphorylation initiates the expression of the lipogenesis-activating transcription factor sterol regulatory element-binding protein 1 (SREBP1). SREBP1 levels were higher after PTEN knockdown and may account for the observed enhanced adipogenesis. To validate this, we overexpressed constitutively active FOXO1 in PTEN CRISPR cells and found reduced adipogenesis, accompanied by SREBP1 downregulation. We observed that PTEN CRISPR cells showed less senescence compared with controls and the senescence marker CDKN1A (p21) was downregulated in PTEN knockdown cells. Cellular senescence was the most significantly enriched pathway found in RNA-Seq of PTEN knockdown versus control cells. These results provide evidence that PTEN is involved in the regulation of APC proliferation, differentiation, and senescence, thereby contributing to aberrant adipose tissue growth in patients with PHTS.

Highlights

  • We observed that lipoma cells from a patient with a phosphatase and tensin homolog (PTEN) germline pathogenic variant retain their differentiation capacity over a prolonged period [13]

  • On the mRNA level, we found a reduction of PTEN expression to 0.33 ± 0.08 fold (p < 0.0001) in visceral (Fig. 1B) and 0.3 ± 0.01 fold (p = 0.009) in subcutaneous stromal vascular fraction (SVF) cells (Fig. S1A)

  • Both obesity and lipoma formation are characterized by adipose tissue overgrowth

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Summary

RESEARCH ARTICLE

PTEN regulates adipose progenitor cell growth, differentiation, and replicative aging. Adipose progenitor cells (APCs) lose their capacity to differentiate into adipocytes during continuous culture, whereas APCs from lipomas of patients with PHTS retain their adipogenic potential over a prolonged period. It remains unclear which mechanisms trigger this aberrant adipose tissue growth. Cellular senescence was the most significantly enriched pathway found in RNA-Seq of PTEN knockdown versus control cells These results provide evidence that PTEN is involved in the regulation of APC proliferation, differentiation, and senescence, thereby contributing to aberrant adipose tissue growth in patients with PHTS. We thereby observed phenomena associated with proliferation, differentiation, and replicative aging of fat cell progenitors pointing to a role for PTEN in lipoma formation

Results
PTEN downregulation restored adipogenic potential in highpassage SVF cells
PTEN levels were upregulated during cellular aging
Expression changes after PTEN downregulation
Discussion
Cell culture and adipocyte differentiation
PTEN siRNA transfection
Lipid staining
Proliferation assay
Western blot analysis
Immunofluorescence staining
Differential gene expression and gene set enrichment
Statistical analysis
Read preprocessing and mapping
Full Text
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