The differentiation of MSCs into adipocytes is a complex process associated with diverse transcriptional changes [12]. Here, we identified a novel role for LRRC1 as a regulator of this adipogenic differentiation network.
We initially observed dynamic changes in LRRC1 expression levels in the context of adipogenesis. At present, the precise mechanisms governing LRRC1 expression are incompletely understood, with one study of hepatoma cells having shown decreased promoter methylation to contribute to the epigenetic upregulation of this gene in these cells [15]. In non-small cell lung cancer cells, however, LRRC1 expression was reported to be post-translationally regulated by miR-193a produced by bone marrow MSCs [16]. Here, we further found LRRC1 to be under the transcriptional control of PPARγ in the context of adipocytic differentiation. As PPARγ is a transcription factor that is essential to the regulation of adipogenesis, its knockdown can impair this physiological process [17–19]. Mechanistically, PPARγ binds to specific PPAR response element (PPRE) regions within target gene promoters to alter their expression [20]. Certain adipogenesis-associated genes are transcriptionally regulated by PPARγ, such as FATP (fatty acid transport protein)[21], adipocyte fatty acid binding protein (aP2)[22], and lipoprotein lipase (LPL)[23]. Notably, we herein found that while LRRC1 knockdown impaired adipocytic differentiation in MSCs, it had a negligible impact on PPARγ expression, suggesting a lack of feedback regulation between these two factors and underscoring LRRC1 as a secondary mediator of adipogenesis. Moreover, LRRC1 transcription is not solely regulated by PPARγ in this model system, as evidenced by the divergent expression patterns of these two genes on day 7 of the adipogenic process.
LRRC1 is a member of the LAP (leucine-rich repeat and PDZ) family of proteins that was initially identified as a regulator of cellular polarity, cell-cell connections, and oncogenic transformation [24]. Given that a loss of apical-basal polarity is generally related to malignant phenotypic outcomes in epithelial tissues, many studies have examined the oncogenic role of LRRC1. For example, in one report, LRRC1 was found to regulate breast cancer stem cell fate determination [10], while it has also been shown to influence HCC cell growth and colony formation [11], and to contribute to NSCLC cell cisplatin resistance [16]. LRRC1 also functions in non-oncogenic contexts, being expressed, for example, in myotubes wherein it influences the physical dimensions of agrin-dependent AChR aggregates and the density of microclusters formed in the absence of agrin [25]. Together with scribble and Erbin, LRRC1 also exhibits significant accumulation at neuromuscular junction (NMJ) regions in synaptic cells, likely regulating associated morphology and neurotransmission via nicotinic acetylcholine receptor clusters [26]. Moreover, LRRC1 is highly expressed in polarized epithelial tissue in Xenopus laevis embryos during the late stages of development, including the cement gland, eyes, tail bud, branch arcs, and developing otic vesicles [27]. These findings highlight the complex biological roles played by LRRC1.
At present, the signaling pathways engaged downstream of LRRC1 have yet to be fully clarified, although it has been shown to regulate WNT/β-catenin activity. Specifically, the LRRC1 homolog Scrib has been shown to negatively regulate WNT/β-catenin signaling in HEK293 cells [28]. Moreover, in LRRC1-knockout mice, LRRC1-deficient induced higher levels of WNT ligand in breast cancer stem cells [10]. The WNT/β-catenin pathway serves as a key hub for the regulation MSCs adipogenic/osteogenic differentiation [29]. However, our data collected in the context of MSCs adipocytic differentiation did not provide any evidence for the ability of LRRC1 to regulate WNT/β-catenin signaling. The reason may be that we did not choose enough time points for detection. Instead, our proteomic analyses revealed significant changes in the expression of adipogenesis-related genes including Fatty Acid Synthase (FASN, gene ID: 2194), Hormone-Sensitive Lipase (LIPE, gene ID: 3991), and Stearoyl-CoA Desaturase (SCD, gene ID: 6319). FASN is a multifunctional enzyme responsible for catalyzing long-chain saturated fatty acid de novo biosyntehsis from acetyl CoA and malonyl COA when NADPH is available [30], with reduced FASN expression contributing to impaired adipogenesis [31]. LIPE can hydrolyze stored triglycerides in adipose and cardiac tissue to yield free fatty acids, with the dysregulation of its expression similarly contributing to aberrant adipogenic activity [32]. SCD is an iron-containing enzyme that is required for adipogenesis owing to its ability to catalyze a rate-limiting step in unsaturated fatty acid synthesis [33]. The functions of these three proteins are interrelated in the context of adipogenesis. However, the specific mechanisms whereby LRRC1 impacts the expression of these genes remains unclear and warrants further study.
In summary, these results support a model in which LRRC1 is a downstream PPARγ target that regulates the adipocytic differentiation of MSCs. Mechanistically, this regulatory activity may be associated with the control of the expression of adipogenesis-related proteins such as FASN, SCD, or LIPE. Together, these data enrich current understanding regarding the mechanistic basis for adipogenesis while providing a foundation for future functional studies of LRRC1.