Differential expression and accumulation of 14-3-3 paralogs in 3T3-L1 preadipocytes and differentiated cells

Uhart

et al. 2020

Preprint

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Background 14-3-3 protein family binds and regulate hundreds of serine/threonine phosphorylated proteins. Considered as redundant, ubiquitous and constantly expressed, this protein family was treated as an accessory for many signaling systems. Recently, specific biological functions were discovered for each of its seven paralogs in humans. Here, we studied the reversible inhibition by acetylation of its essential N- ε -lysine 49/51 residue during the osteogenic differentiation of human adipose-derived stem cells. Methods Human adipose-derived stem cells were obtained from healthy patients who underwent a dermolipectomy. The material was processed to obtain the ASCs from the stromal vascular fraction. Proteins of interest were analyzed by confocal microscopy and Western blot, and the expression level of selected proteins were modified by using lentivirus and specific shRNAs. Results We found that during the differentiation of ASC, the levels of 14-3-3 acK49/51 increased, showing that inhibition of 14-3-3 is necessary during this process. Among the 7 paralogs of this family, the inhibition by this posttranslational modification occurs mostly on the paralog β , located specifically in the nucleus, where 14-3-3 was described to bind to H3 histone and many transcription factors. Paralog specific inhibition by short hairpin RNA showed that silencing of 14-3-3 β gene, but not 14-3-3γ, increases significantly the osteogenic potential of the cells. Transdifferentiation from osteogenic to adipogenic linage of cell lines that were specifically silenced for 14-3-3 β o γ paralogs showed that 14-3-3b could be a negative regulator of the differentiation to the osteogenic lineage. Conclusion We showed that specifically 14-3-3 β is a negative regulator of osteogenesis. The levels of its inhibition by acetylation on lysine 51 is the key cellular mechanism to regulate it. The HBO1 acetyltransferase could be the enzyme responsible of this modification. Cells were this inhibition was emulated by decreasing the levels of expression of 14-3-3 β showed significant increased potential for the differentiation to the osteogenic lineage.

Section: -3-3 Paralogs Are Regulated Differently During Asc Osteogementioning

confidence: 54%

Section: -3-3 Paralogs Are Regulated Differently During Asc Osteogementioning

confidence: 99%

Section: -3-3 Paralog B -But Not G-is Regulated By Acetylation In Lmentioning

confidence: 99%

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14-3-3β paralog is inhibited by acetylation during differentiation to the osteogenic lineage.

López

Uhart

et al. 2020

Preprint

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“…The adipogenic potential of hASCs tends to decline with consecutive passages under standard culture conditions (Li et al, 2017 ). To induce this process in vitro , we used low passage (up to 6) hASCs with an optimized drug cocktail (Gojanovich et al, 2016 ), based on a comparison of previously used formulations (Gimble and Guilak, 2003 ; Lee et al, 2004 ; Rodriguez et al, 2004 ; Dicker et al, 2005 ; Bunnell et al, 2008 ; Zhu et al, 2008 ; da Silva Meirelles and Covas, 2011 ; Kim et al, 2016 ). These include DMEM (high or low glucose), 10% v/v FBS, and variable concentrations of Insulin, Dexamethasone, Indomethacin, or Rosiglitazone, with or without 0.5 mM IBMX.…”

Section: Anticipated Resultsmentioning

confidence: 99%

Human Adipose-Derived Mesenchymal Stem/Stromal Cells Handling Protocols. Lipid Droplets and Proteins Double-Staining

Giménez

Masone

et al. 2018

Front. Cell Dev. Biol.

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Human Adipose-derived mesenchymal stem/stromal cells (hASCs) are of great interest because of their potential for therapeutic approaches. The method described here covers every single step necessary for hASCs isolation from subcutaneous abdominal adipose tissue, multicolor phenotyping by flow cytometry, and quantitative determination of adipogenic differentiation status by means of lipid droplets (LDs) accumulation, and Western blot analysis. Moreover, to simultaneously analyze both LDs accumulation and cellular proteins localization by fluorescence microscopy, we combined Oil Red O (ORO) staining with immunofluorescence detection. For LDs quantification we wrote a program for automatic ORO-stained digital image processing implemented in Octave, a freely available software package. Our method is based on the use of the traditional low cost neutral lipids dye ORO, which can be imaged both by bright-field and fluorescence microscopy. The utilization of ORO instead of other more expensive lipid-specific dyes, together with the fact that the whole method has been designed employing cost-effective culture reagents (standard culture medium and serum), makes it affordable for tight-budget research laboratories. These may be replaced, if necessary or desired, by defined xeno-free reagents for clinical research and applications.

“…Previous results from our laboratory showed that during the adipogenic differentiation of NIH3T3-L1 preadipocytes cell line, the expression levels of 14-3-3 protein family isoforms are independently regulated. 29 As it has been proposed that adipogenic and osteoblastic differentiation are highly related processes, we analyzed the levels of six 14-3-3 isoforms in ASC treated with ODM (for composition, see Section 2). We isolated ASC from healthy patients who underwent a dermolipectomy surgery and voluntarily donated abdominal subcutaneous adipose tissue after signing bioethics committee-approved informed consent documents.…”

Section: -3-3 Isoforms Levels Are Regulated Differently During Asc Osteogenic Differentiationmentioning

confidence: 99%

14‐3‐3β isoform is specifically acetylated at Lys51 during differentiation to the osteogenic lineage

Frontini-López

J of Cellular Biochemistry

Veliz

et al. 2021

Self Cite

The 14-3-3 protein family binds and regulates hundreds of serine/threonine phosphorylated proteins as an essential component of many signaling networks. Specific biological functions are currently been discovered for each of its seven isoforms in mammals. These proteins have been traditionally considered unregulated; however, its acetylation in an essential lysine residue, causing its inactivation, was recently published. Here, we studied the acetylation state of this lysine 49/51 during the osteogenic differentiation of human adipose-derived stem cells. We found that during this process, the levels of 14-3-3β (but not its isoform 14-3-3γ) acK49/51 increase, representing the first report linking this PTM to a specific isoform and a cellular process. Our results suggested that this posttranslational modification could be catalyzed by the HBO1 acetyltransferase, as overexpression of HBO1 increased specifically 14-3-3 acK49/51 acetylation. Acetylated 14-3-3 proteins are located primarily in the nucleus, where their active state has been described to bind H3 histones and many transcription factors. The inhibition of the expression of different isoforms showed that the specific silencing of the 14-3-3β gene, but not γ, increased significantly the osteogenic potential of the cells. This result correlated to the increase in acetylation of 14-3-3β Lys 49/51 during osteogenesis. The possible role of this PTM in osteogenesis is discussed.