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ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells

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Abstract

Mesenchymal stem cells (MSCs) are multipotent progenitors present in the bone marrow stroma and in subcutaneous abdominal fat, an abundant and easily accessible source of MSCs with the ability to differentiate along multiple lineage pathways. The stem cell-associated transcription co-factor Zinc Finger Protein 521 (ZNF521/zfp521) has been implicated in the control of the homeostasis of hematopoietic, neural and osteo-adipogenic progenitors. Here we document through the analysis of a panel of human adipose-derived stem cells (hADSCs), that ZNF521 strongly inhibits the generation of mature adipocytes. Enforced overexpression of ZNF521 in these cells resulted in a significant delay and reduction in adipocyte differentiation upon exposure to inducers of adipogenesis. Of particular relevance, ZNF521 was able to inhibit the expression of ZNF423, recently identified as an essential commitment factor necessary for the generation of pre-adipocytes. Conversely, silencing of ZNF521 was found to significantly enhance the adipogenic differentiation of hADSCs. Inhibition of adipogenesis by ZNF521 was at least in part due to inhibition of EBF1. Taken together, these results confirm a role for ZNF521 as a key negative regulator of adipocyte differentiation of hADSCs.

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Abbreviations

ZNF521:

Zinc finger protein 521

MSCs:

Mesenchymal Stem Cells

hADSCs:

human Adipose-Derived Stem Cells

ZNF423:

Zinc finger protein 423

EBF-1:

Early B Cell Factor-1

PPARγ :

Peroxisome Proliferator-Activated Receptor gamma

C/EBPs :

CAATT/enhancer binding proteins

EBF-2:

Early B Cell Factor-2

ZNF638:

Zinc finger protein 638

NF1 :

Neurofibromin 1

ADD1:

Adipocyte Determination and Differentiation-dependent factor 1

SREBP1:

Sterol Regulatory Element-binding Protein 1

CREB:

Cyclic AMP Response Element-binding protein

KLFs:

Kruppel-like Factors

GATA:

GATA binding protein

PREF-1:

Preadipocyte Factor-1

SIRT1:

Histone deacetylase Sirtuin 1

TAZ:

Transcriptional-coactivator with PDZ-binding motif

Krox20:

EGR C2H2-type zinc-finger protein

FABP4:

Fatty Acid Binding Protein 4

FAS:

Fatty Acid Synthase

LPL:

Lipoprotein Lipase

SCD:

Stearyl-CoA-Desaturase

GLUT-4:

Glucose transporter 4

C2H2-ZF:

Cys2-His2 zinc fingers

WISP2:

WNT1 Inducible Signaling Pathway Protein 2

BMP4 :

Bone Morphogenetic Protein 4

UBC:

Ubiquitin C

IRES:

Intra-Ribosomal Entry Site

eGFP:

Enhanced Green Fluorescent Protein

LDs:

Lipid Droplets

PPRE:

PPAR-Responsive Element

ACOX1:

Acyl-CoA Oxidase 1

DEX:

Dexamethasone

IBTX:

Isobutyl-methylxanthine

BmMSCs:

Bone marrow Mesenchymal Stem Cells

Runx2 :

Runt-related transcription factor 2

Smad:

Small mother against decapentaplegic

NuRD:

Nucleosome Remodeling Deacetylase

PDGFR-α:

Platelet-Derived Growth Factor Receptor –α

HDAC:

Histone Deacetylase

GAPDH:

Glyceraldehyde 3-Phosphate Dehydrogenase

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Acknowledgements

All authors are acknowledged for their contribution to the study.

Funding

This work was supported by funds from PON03PE_00009_2 ICaRe and PON01_02834 PROMETEO to GM. AA, GN, BC, VL, YM were supported by PhD Programme in Molecular and Translational Oncology and Innovative Surgical Medical Technologies. EC and SS were supported by fellowship to fund PON03PE_00009_2 ICaRe.

Author information

Author notes

  1. Emanuela Chiarella, Annamaria Aloisio, Heather Mandy Bond, Giovanni Morrone contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Molecular Haematopoiesis and Stem Cell Biology, University Magna Græcia, Catanzaro, Italy

    Emanuela Chiarella, Annamaria Aloisio, Stefania Scicchitano, Valeria Lucchino, Ylenia Montalcini, Maria Mesuraca, Heather Mandy Bond & Giovanni Morrone

  2. Tecnologica Research Institute- Marrelli Hospital, Crotone, Italy

    Bruna Codispoti

  3. UCSF Hellen Diller Cancer Center, University of California, San Francisco, CA, USA

    Giovanna Nappo

  4. Federal University of Paraíba (UFPB), João Pessoa, Brazil

    Augusto Camarotti

  5. Department of Orthopaedic & Trauma Surgery, University “Magna Graecia”, Catanzaro, Italy

    Olimpio Galasso & Giorgio Gasparini

  6. Department of Plastic Surgery, University “Magna Graecia”, Catanzaro, Italy

    Manfredi Greco

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  1. Emanuela Chiarella
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Contributions

GM and HMB conceived and designed the experiments; EC, AA, GN performed the experiments and SS, VL, YM, BC and AC analyzed the data; GM, HBM, EC, AA, MM, wrote the paper. MG, OG, GG helped to draft the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Emanuela Chiarella.

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Electronic supplementary material

Additional file 1A-1B

In a set of six different hADSCs experiments (n = 4) of ZNF521 overexpression and subsequent adipocyte differentiation the mRNA levels of the main adipocyte markers are consistantly reduced. (PNG 432 kb)

High resolution image (TIF 2543 kb)

Additional file 1B

(PNG 178 kb)

High resolution image (TIF 950 kb)

Additional file 2A-2B

Six different experiments of ZNF521 silencing (n = 4) followed by adipocyte differentiation demonstrate a significant increase of the main genes involved during adipocyte differentiation. Only in one of these experiments (N°6) the shRNA-2 was inefficiently and consequently the induction of adipogenesis was only minimum. (PNG 1142 kb)

High resolution image (TIF 3841 kb)

Additional file 2B

(PNG 367 kb)

High resolution image (TIF 1197 kb)

Additional file 3

The ImageJ based programme used to process three images for each sample and to obtain the percentage of Oil red O stained area for field and the red intensity (pixels). The mean of percentage of stained area and the mean of absolute red intensity for each sample were compared to the corresponding controls both in stably overexpressing (Additional Fig. 3A) and silencing experiments (Additional Fig. 3B). (PNG 26 kb)

High resolution image (TIF 319 kb)

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Chiarella, E., Aloisio, A., Codispoti, B. et al. ZNF521 Has an Inhibitory Effect on the Adipogenic Differentiation of Human Adipose-Derived Mesenchymal Stem Cells. Stem Cell Rev and Rep 14, 901–914 (2018). https://doi.org/10.1007/s12015-018-9830-0

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