Dataset Information

Early adipogenesis is repressed through the newly identified FHL2-NFAT5 signaling complex

ABSTRACT: The LIM-domain-only protein FHL2 is a modulator of signal transduction and has been shown to direct the differentiation of mesenchymal stem cells toward osteoblasts and myocytes phenotypes. We hypothesized that FHL2 may simultaneously interfere with the induction of the adipocyte lineage. Therefore, we investigated the role of FHL2 in adipocyte differentiation using pre-adipocytes isolated from mouse adipose tissue and the 3T3-L1 (pre)adipocyte cell line. Here we report that FHL2 is expressed in pre-adipocytes and for accurate adipocyte differentiation, this protein needs to be downregulated during the early stages of adipogenesis. More specifically, constitutive overexpression of FHL2 drastically inhibits adipocyte differentiation in 3T3-L1 cells, which was demonstrated by suppressed activation of the adipogenic gene expression program as shown by extensive RNAseq analyses, and diminished lipid accumulation. To identify the protein-protein interactions mediating this repressive activity of FHL2 on adipogenesis, we performed affinity-purification mass spectrometry (AP-MS). This analysis revealed the interaction of FHL2 with the Nuclear factor of activated T-cells 5 (NFAT5), an established inhibitor of adipocyte differentiation. NFAT5 knockdown rescued the inhibitory effect of FHL2 overexpression on 3T3-L1 differentiation, indicating that these proteins act cooperatively. In conclusion, we present a new regulatory function of FHL2 in early adipocyte differentiation and revealed that FHL2-mediated inhibition of pre-adipocyte differentiation is dependent on its interaction with NFAT5.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Preadipocyte

SUBMITTER: Harmjan Vos

LAB HEAD: Carlie J.M. de Vries

PROVIDER: PXD033974 | Pride | 2023-11-13

REPOSITORIES: Pride

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			Action	DRS
	20201016_E1_FM_75_RM_MHQ_0351_FhL2_1.raw	Raw
	20201016_E1_FM_75_RM_MHQ_0351_FhL2_1_FS45.raw	Raw
	20201016_E1_FM_75_RM_MHQ_0351_FhL2_1_FS65.raw	Raw
	20201016_E1_FM_75_RM_MHQ_0351_FhL2_2.raw	Raw
	20201016_E1_FM_75_RM_MHQ_0351_FhL2_2_FS45.raw	Raw

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Early adipogenesis is repressed through the newly identified FHL2-NFAT5 signaling complex.

Clemente-Olivo Maria P MP Hernández-Quiles Miguel M Sparrius Rinske R van der Stoel Miesje M MM Janssen Vera V Habibe Jayron J JJ van den Burg Janny J Jongejan Aldo A Alcaraz-Sobrevals Paula P van Es Robert R Vos Harmjan H Kalkhoven Eric E de Vries Carlie J M CJM

Cellular signalling 20230105

The LIM-domain-only protein FHL2 is a modulator of signal transduction and has been shown to direct the differentiation of mesenchymal stem cells towards osteoblast and myocyte phenotypes. We hypothesized that FHL2 may simultaneously interfere with the induction of the adipocyte lineage. Therefore, we investigated the role of FHL2 in adipocyte differentiation. For these studies pre-adipocytes isolated from mouse adipose tissue and the 3T3-L1 (pre)adipocyte cell line were applied. We performed FH ...[more]

PMID: 36610523

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Project description:Visceral fat (VF) and subcutaneous fat (SF) are developmentally different tissues with different gene expression. Islet-1 (ISL1), a LIM-homeobox transcription factor with important developmental and regulatory function in islet, neural, and cardiac tissue, is virtually absent in SF but substantially expressed in the stromovascular [preadipocyte containing] fraction of VF; expression correlates negatively with adiposity in rodents and man. ISL1 expression is transiently increased in 3T3-L1 preadipocytes during early differentiation, suggesting a functional role. To examine the role of ISL1 in adipogenesis, we tested whether retroviral overexpression of ISL1 in 3T3-L1 preadipocytes affected their ability to differentiate into mature adipocytes. Terminal differentiation was assessed by Oil Red O [lipid droplet] staining and by immunoblot detection of adipocyte marker proteins, including aP2 and GLUT4. ISL1 significantly inhibited lipid droplet formation, reduced lipid accumulation (about 80% inhibition, p<0.05), and substantially inhibited aP2 and GLUT4 expression. ISL1 did not inhibit expression of C/EBPb and C/EBPd after induction of differentiation, but reduced PPARg and C/EBPa by >50% at both mRNA and protein level. In addition, the PPARg agonist, rosiglitazone, substantially rescued ISL1 inhibited adipogenesis in the absence of exogenous PPARg, and fully rescued in the presence of exogenous PPARg. In summary, ISL1 overexpression inhibited fat droplet formation, lipid accumulation, and adipocyte-specific gene expression; there was accompanying inhibition of C/EBPa, PPARg and downstream gene expression. We conclude that ISL1 overexpression inhibited adipocyte differentiation by inhibition of PPARg regulated gene expression. As abdominal obesity strongly correlates with insulin resistance, and cardiovascular risk, ISL1 up-regulation may impact abdominal obesity and its concomitant metabolic derangements. Total cellular RNA was isolated from 3T3-L1 cells expressing Flag-ISL1 or not at 48 h following treatment with differentiation cocktail. Individual RNA from biological triplicates was used for microarray analysis.