Elucidation of the 14-3-3ζ interactome reveals critical roles of RNA splicing factors during adipogenesis

Abstract

Adipogenesis is facilitated by a complex signaling network requiring strict temporal and spatial organization of effector molecules. Molecular scaffolds, such as 14-3-3 proteins, coordinate such events, and we have previously identified 14-3-3ζ as an essential scaffold in adipocyte differentiation. The interactome of 14-3-3ζ is large and diverse, and it is possible that novel adipogenic factors may be present within it. Mouse embryonic fibroblasts from mice over-expressing a TAP-epitope-tagged 14-3-3ζ molecule were generated, and following the induction of adipogenesis, TAP-14-3-3ζ complexes were purified, followed by mass spectrometry analysis to determine the 14-3-3ζ interactome. Over 100 proteins were identified as being unique to adipocyte differentiation, of which 56 were novel interacting partners. Previously established regulators of adipogenesis (ie, Ptrf/Cavin1 and Phb2) were found within the 14-3-3ζ interactome, confirming the ability of this approach to identify regulators of adipocyte differentiation. An enrichment of proteins in the interactome related to RNA metabolism, processing, and splicing was identified, and analysis of transcriptomic data revealed that 14-3-3ζ depletion in 3T3-L1 cells affected the alternative splicing of mRNA during adipocyte differentiation. Of the RNA splicing factors within the 14-3-3ζ interactome, depletion of Hnrnpf, Hnrnpk, Ddx6, and Sfpq by siRNA revealed essential roles of these proteins in adipogenesis and their roles in the alternative splicing of Lpin1. In summary, novel adipogenic factors can be detected within the 14-3-3ζ interactome, and further characterization of additional proteins within the 14-3-3ζ interactome has the potential of identifying novel targets to block the expansion of adipose tissue mass that occurs in obesity.

Reducing 14-3-3ζ expression influences adipocyte maturity and impairs function

“…We previously reported essential roles of one of the seven mammalian isoforms, 14-3-3ζ, in adipogenesis, as systemic deletion of 14-3-3z in mice resulted in significant reductions in visceral adipose tissue mass and decreased expression of mature adipocyte markers in gonadal adipocytes (34). Furthermore, through the use of proteomics to elucidate the 14-3-3z interactome, we found that 14-3-3z is necessary for RNA splicing during adipocyte differentiation (42). However, whether 14-3-3ζ can also influence metabolic processes specific to mature adipocytes has not been fully examined.…”

“…3T3-L1 cells were maintained in DMEM (Life Technologies Corporation, Grand Island, NY), supplemented with 10% newborn calf serum (NBCS) and 1% penicillin/streptomycin (P/S) and were seeded onto 12-well plates (100,000 cells/well) or 10cm dishes (2x10 6 cells/dish) 2 days prior to the induction of differentiation. Confluent cells were treated with a differentiation cocktail (DMEM supplemented with 10% FBS, 1% P/S, 500 μM IBMX, 500 nM dexamethasone and 172 nM insulin) for 48 hours, followed by media replacement (DMEM with 10% FBS, 1% P/S and 172 nM insulin) every 2 days for 7-8 days (34,42). Knockdown and overexpression of 14-3-3ζ was achieved by transfecting day 7-8 differentiated cells with scrambled control siRNA, 14-3-3ζ-specific siRNA (Ambion, Austin, TX), GFP control plasmids, or plasmids encoding 14-3-3ζ (14-3-3ζ IRES-GFP) using the Amaxa Cell Line Nucleofector Kit L, as per manufacturer's instructions (Lonza, Koln, Germany).…”

“…Knockdown and overexpression of 14-3-3ζ was achieved by transfecting day 7-8 differentiated cells with scrambled control siRNA, 14-3-3ζ-specific siRNA (Ambion, Austin, TX), GFP control plasmids, or plasmids encoding 14-3-3ζ (14-3-3ζ IRES-GFP) using the Amaxa Cell Line Nucleofector Kit L, as per manufacturer's instructions (Lonza, Koln, Germany). Differentiated 3T3-L1 cells were stained with Oil Red-O (ORO) and measured, as previously described (34,42). All studies with 3T3-L1 cells were performed on cells between passages 10-16.…”

“…Measurements of mRNA were performed with SYBR green chemistry using the QuantStudio 6-flex Real-time PCR System (ThermoFisher Scientific). All data were normalized to either Hprt or Actinb by the ΔC(t) method (34,42). Primer sequences are listed in Supplemental Table 1.…”

“…Differentiated 3T3-L1 cells were lysed in RIPA buffer (0.9% NaCl, 1% v/v triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, and 0.6% tris base) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich). Proteins were resolved by SDS-PAGE and transferred to PVDF membranes, as previously described (34,42). Gonadal and inguinal adipose tissues were harvested from WT and adi14-3-3zKO mice, fixed in 4% paraformaldehyde (Sigma-Aldrich), embedded in paraffin, and sectioned to 6 µm thickness.…”