HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Mol Cell Manuscript Author Biochem. Author Manuscript Author manuscript; available in PMC 2019 November 01. Published in final edited form as: Mol Cell Biochem. 2018 November ; 448(1-2): 299–309. doi:10.1007/s11010-018-3334-8. MCAM knockdown impairs PPARγ expression and 3T3-L1 fibroblasts differentiation to adipocytes Matías Gabrielli1,2, Damián G. Romero3, Claudia N. Martini1, Laura Judith Raiger Iustman1,2, and María del C. Vila1,2,° 1Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Buenos Aires, Argentina. 2Universidad de Buenos Aires. CONICET. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales (IQUIBICEN). Buenos Aires, Argentina. 3Department of Cell and Molecular Biology, University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, USA Abstract We investigated for the first time the expression of MCAM (Melanoma cell adhesion molecule) and its involvement in the differentiation of 3T3-L1 fibroblasts to adipocytes. We found that MCAM mRNA increased subsequent to the activation of the master regulator of adipogenesis, PPARγ, and this increase was maintained in the mature adipocytes. On the other hand, MCAM knockdown impaired differentiation and induction of PPARγ as well as expression of genes activated by PPARγ. However, events that precede and are necessary for early PPARγ activation such as C/EBPβ induction, β-catenin downregulation and ERK activation, were not affected in the MCAM knockdown cells. In keeping with this, the increase in PPARγ mRNA that precedes MCAM induction was not altered in the knockdown cells. In conclusion, our findings suggest that MCAM is a gene upregulated and involved in maintaining PPARγ induction in the late but not in the early stages of 3T3-L1 fibroblasts adipogenesis. Keywords MCAM/CD146; 3T3-L1 fibroblasts; adipogenesis; PPAR gamma; C/EBP beta INTRODUCTION Obesity is a frequent health problem due to an increase in the number (hyperplasia) and size (hypertrophy) of adipocytes which is associated with metabolic syndrome, type 2 diabetes mellitus, hypertension and cardiovascular disease [1, 2]. Adipocytes are derived from mesenchymal stromal cells, these are precursor cells which are able to differentiate to °Corresponding Author: María del Carmen Vila, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, 1428, Buenos Aires, Argentina. Phone 541145763300 Ext: 443, Fax: 541145763342, [email protected]. CONFLICTS OF INTEREST The authors declare that there are no conflicts of interest. Gabrielli et al. Page 2 several lineages such as: osteocytes, chondrocytes and adipocytes. The knowledge of the Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author signaling involved in the development of adipocytes may help in the control of obesity and also may be useful to understand how to select a specific cell fate in the case of tissue regeneration therapy [3]. Adipogenesis is a coordinated and complex process that starts with the upregulation of two early transcription factors CCAAT/enhancer-binding protein beta (C/EBPβ) and C/EBPδ and then they induce the expression of Peroxisome proliferator-activated receptor gamma (PPARγ), the master gene in adipogenesis which, in turn, increases the expression of proteins associated with the adipocyte phenotype [4]. Accordingly, a dominant-negative PPARγ mutant inhibits adipogenesis [5, 6]. 3T3-L1 fibroblasts are precursor cells used as a model to study adipogenesis. These preadipocytes are induced to differentiate with a mixture containing insulin, dexamethasone and methylisobutylxanthine. A few days later, cells become round and start to accumulate lipid droplets and terminal differentiation is attained 7 to 10 days after induction. In order to identify genes relevant for adipogenesis, we have previously performed a microarray analysis of genes regulated during differentiation of 3T3-L1 fibroblasts [7]. In this microarray, among the genes that were upregulated, we found genes known to be involved in adipogenesis and also identified the Mcam gene. We decided to analyze MCAM as little is known about its regulation and role during adipocyte differentiation. MCAM (melanoma cell adhesion molecule) also known as CD146, is an integral membrane protein belonging to immunoglobulin (Ig) superfamily of cell adhesion molecules. It was originally identified as a marker associated to tumor progression and metastasis in human melanoma, but later was found to be present in other malignant lesions as well as in normal tissues [8, 9]. MCAM is also a mesenchymal stromal cell (MSC) marker whose expression positively correlates with the differentiation potential [10]. The protein structure is composed of an extracellular region with five Ig-like domains (V-V-C2-C2-C2), a transmembrane region and a cytoplasmic domain and it is conserved among human, mouse and other organisms [9]. MCAM functions as a cell surface receptor that signals into the cell upon binding of different ligands. Laminin-411, Wnt5a, netrin-1 has been described as ligands for MCAM [11–13]. In addition, MCAM has been proposed as a co-receptor for VEGF-R2 and is involved in VEGF signaling through AKT/p38 MAPKs/NF-κB to promote cell migration and angiogenesis in endothelial cells [14]. It has also been shown that upon its engagement in endothelial cells, MCAM recruits and activates Fyn kinase leading to a protein kinase cascade that has been proposed to be linked to actin cytoskeleton remodeling [15]. In this paper, we investigate the expression of MCAM and its involvement in 3T3-L1 fibroblasts differentiation to adipocytes. Mol Cell Biochem. Author manuscript; available in PMC 2019 November 01. Gabrielli et al. Page 3 Author ManuscriptAuthor MATERIALS Manuscript Author AND Manuscript Author METHODS Manuscript Author Reagents Dulbecco’s modified Eagle’s medium (DMEM), antibiotics, trypsin, insulin, Bodipy 493/503 and Lipofectamine 2000 were obtained from Invitrogen (Carlsbad, CA, USA). Methylisobutylxanthine, dexamethasone were obtained from Sigma-Aldrich (St. Louis, MO, USA). Puromycin was purchased from Invivogen (San Diego, CA, USA). 3T3-L1 fibroblasts were obtained from Asociación Banco Argentino de Células (origin: ATCC), NIH 3T3 from ATCC and HEK293FT from Invitrogen. 3T3-L1 cells could not be authenticated due to the lack of an STR DNA profile database for mouse cells. However, the adipocyte phenotype that we observed only after differentiation is consistent with the fact that the cells used were indeed 3T3-L1 fibroblasts. Fetal Bovine Serum (FBS) was purchased from Natocor (Argentina). Cell culture and differentiation induction 3T3-L1 and NIH 3T3 fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 0.1 mg/mL streptomycin, 100 U/mL penicillin and 250 ng/mL fungizone (DMEM + 10% FBS). In differentiation experiments, plated cells were allowed to reach confluence and two days later differentiation was induced with a mixture containing: 10 μg/mL insulin (I), 0.1 μM dexamethasone (D), and 0.5 mM methylisobutylxanthine (M) in DMEM with 10% FBS (IDM). After 3 days medium was replaced with DMEM + 10% FBS supplemented with 10 μg/mL insulin and cells were fed every 2 days thereafter with DMEM + 10% FBS. In some experiments, 2-day postconfluent 3T3-L1 fibroblasts were stimulated with individual inducers (I, D, M) or combinations of two inducers (ID, DM, IM) at the concentrations indicated above. After 3 days culture medium was replaced with DMEM + 10% FBS alone or supplemented with 10 μg/mL insulin in those assays induced in the presence of insulin. Construction of lentiviral vectors, virus production and transduction of 3T3-L1 fibroblasts A fragment coding a small hairpin RNA (shRNA) that targeted murine Mcam was subcloned into the pLKO.1 plasmid [16] according to the protocol available from Addgene (http:// www.addgene.org/tools/protocols/plko/). The shRNA sequence 5’- CCGACTCGTAAGAGTGAATTT-3’, which targets the coding DNA sequence (CDS) of the murine Mcam mRNA, was selected from the TRC RNAi Consortium library (http:// www.broad.mit.edu/genome_bio/trc/rnai.html), clone ID TRCN0000113082. The empty pLKO.1 vector, without the shRNA construct and the stuffer sequence, was used as a negative control. For lentivirus production, exponentially growing HEK293FT cells cultured in 6-well plates were transfected in 1.5 ml of medium using 6 μl of Lipofectamine 2000 with 1.2 μg of either the shRNA containing vector or the empty vector, in combination with 1 μg of the pCMV- ΔR8.9 packaging plasmid and 0.3 μg of the pCMV-VSVG envelope plasmid. Media was Mol Cell Biochem. Author manuscript; available in PMC 2019 November 01. Gabrielli et al. Page 4 replaced with fresh one after 6 hours. Lentivirus containing media were harvested at 48 h Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author and 72 h after transfection and filtered through a 0.45 μm filter. To establish cell lines stably expressing shRNA or control vector, exponentially growing 3T3-L1 fibroblasts in 6-well plates were transduced with virus-containing media supplemented with 8 μg/mL polybrene. After 24 h, 3T3-L1 cells were transduced again with lentivirus containing media for another 24 h. Then, transduced cells were subjected to selection with culture media supplemented with 5 μg/mL puromycin for 4–6 days. Oil-Red-O