Protein kinase CK2 in adipogenesis and in adipocyte metabolism

Obesity is a multifactorial, chronic, relapsing, progressive disease marked by abnormal and/or excessive accumulation of adipose tissue (AT) that presents a risk for health. AT is a dynamic and plastic endocrine organ able to rapidly expand by both increase in size of existing adipocytes (hypertrophy) and formation of new adipocytes through differentiation of resident precursors (hyperplasia). We previously identified CK2 up-regulation as a hallmark of AT pathological expansion in patients with obesity and diabetes. CK2 is a constitutively active Ser/Thr protein kinase, composed of 2 catalytic (α/α’) and 2 regulatory (β) subunits, which has already been studied in the adipogenic differentiation of human mesenchymal stem cells, where its expression and activity are high at the beginning of the process and decrease in mature fat cells. In this thesis, we aim to deeply characterize the role of CK2 and its subunits in adipogenesis, as well as CK2 involvement in the regulation of adipocyte metabolism. We exploited a double approach of pharmacological and genetic targeting of CK2 using the 3T3-L1 murine preadipocyte cell line as standard model of in vitro adipogenesis. In particular, we inhibited CK2 activity performing cell treatments with the specific CK2 inhibitors SGC-CK2-1 and CX-4945, and we targeted CK2 subunits by RNA interference (RNAi) or CRISPR/Cas9 technologies. The effects on the adipogenic differentiation, induced using standard adipogenic medium (MAD) or MAD supplemented with rosiglitazone (MIR), were evaluated by morphological analysis and Oil-Red-O (ORO) staining. CK2 inhibitors-treated cells, and CK2α and β silenced cells display a slower adipogenic potential upon standard adipogenic induction, thus supporting the role of CK2 in adipogenesis and suggesting a specific function of the  regulatory subunit. To better analyze the role of CK2β subunit, we generated, by CRISPR-Cas9 technology and FACS sorting, 6 3T3-L1 preadipocyte CK2β Knockout (KO) clones characterized by the almost complete ablation of CK2β and the resulting reduction of CK2-specific activity. The majority of CK2β KO clones showed proliferation reduction, morphological changes and were unable to differentiate into mature adipocytes in standard adipogenic conditions. However, induction performed adding rosiglitazone, an anti-diabetic drug and ligand of PPARγ, the master regulator of adipogenic signaling cascade, leads to a partial rescue of adipogenesis. To identify pathways affected by CK2β loss during early adipogenesis we performed transcriptomic analysis at day 0 and day 3 of adipogenic induction. CK2β KO clones exhibited higher expression of genes involved in extracellular matrix deposition and fibrosis and lower expression of adipogenic factors and genes involved in cell cycle and mitosis, rather than controls. Obtained results were validated performing time course qPCR analysis and immunofluorescence experiments, supporting the fundamental role for CK2β regulatory subunit in the early phase of adipogenesis and in matrix/fibrosis remodeling. In addition, to detail the role of CK2 in specific metabolic pathways of mature fat cells, we treated fully differentiated 3T3-L1 adipocytes with the CK2 inhibitors SGC-CK2-1 and CX-4945 and we analyzed polar metabolites and lipids by liquid chromatography/mass spectrometry (LC/MS). CK2 inhibition in mature adipocytes induces accumulation of Branched Chain Amino Acids (BCAAs) and Phosphatidylcholines (PCs), which could associate with insulin resistance conditions. By contrast, CK2 activity could improve insulin sensitivity and exert anti-inflammatory effects, as indicated by carnitine and Fatty Acid esters of Hydroxy Fatty Acids (FAHFAs) accumulation in the absence of CK2 inhibition. In conclusion, our results suggest that targeting of CK2, particularly its β regulatory subunit, may constitute a promising opportunity to develop novel therapies to treat obesity and metabolic disorders.