Protein kinase C delta (PKCδ) splice variant modulates senescence via hTERT in adipose-derived stem cells

Abstract

Background: Adipose-derived stem cells (ADSC) were isolated and characterized from lean and obese subjects. We previously reported that distinct differences were observed in differentiating lean and obese preadipocytes. Protein kinase C delta (PKCδ) is alternatively spliced and has important roles in apoptosis. PKCδI promotes apoptosis and PKCδVIII promotes survival. Our previous data indicated an increase in the survival kinase, PKCδVIII in ADSC derived from an obese donor. We also determined that obese adipocytes were resistant to apoptosis. Here, we determine the relationship between a survival kinase PKCδVIII and hTERT expression in adipose derived stem cells from a lean and obese subject.

Methods: We evaluated the telomerase activity and human telomerase reverse transcriptase (hTERT) expression in lean and obese ADSC. The lean and obese ADSC were purchased as cryopreserved cells from ZenBio™ (Research Triangle Park, NC, USA). Analyses were performed using PRISM™ software and analyzed using two-tailed Student's t-test.

Results: We observed an increase in telomerase in differentiating obese ADSC using western blot analysis. We determined the levels of hTERT splice variants. hTERT α+/β+ splice variant was increased after transfected of PKCδVIII. We next determined whether PKCδVIII over-expression affected the levels of telomerase. The results indicate an increase in telomerase with PKCδVIII over-expression.

Conclusions: Over-expression of PKCδVIII in lean ADSC substantially increased expression of hTERT and telomerase. The decreased senescence seen in obese ADSC may in part be attributed to PKCδVIII. Obese ADSC undergo lower senescence and may have increased growth potential. These results propose a larger epigenetic modification in obese ADSC compared to lean ADSC.

Keywords: Adipose-derived stem cells (ADSC); alternative splicing; human telomerase reverse transcriptase (hTERT); obesity; protein kinase C delta (PKCδ).

Figure 1

High telomerase levels are observed in obese ADSC. Subcutaneous lean or obese ADSC were differentiated in vitro and whole cell lysates were collected on days 0, 4 and 8. (A) Western blot analysis was performed and immunoblotted using telomerase antibody or PKCδVIII-specific antibody. The blots are representative of three experiments performed individually with similar results; (B) Graph represents telomerase or PKCδVIII densitometric units normalized to β-actin and is representative of mean ± SD in four experiments performed independently. Statistical analysis performed by two-tail Student’s t-test; ***P<0.001 highly significant for telomerase between days 0, 4, 8 of lean and obese. ADSC, adipose-derived stem cells; PKCδ, protein kinase C delta.

Figure 2

TRAPeze assay: estimation of processivity for telomerase length. Assay was performed with subcutaneous lean (L) or obese (O) adipose-derived stem cells (ADSC) using either 5 or 3 µg of starting cell extract. Samples were heat inactivated as indicated for controls. TSR8 was used as control template for positive control (Pos) and heat inactivated TSR8 was used as negative control (Neg). The 36 bp internal standard was detected in all samples. The bands indicate telomeric repeats. The assay was repeated four times with similar results.

Figure 3

hTERT splice variants expression in obese ADSC. (A) Subcutaneous lean or obese ADSC were differentiated in vitro. In separate wells, PKCδVIII was transiently transfected on day 2 of differentiation in lean ADSC and then cells were differentiated. RNA was collected from samples on days 0, 4 and 8 (D0, D4, D8) of differentiation. qPCR was performed using hTERT or PKCδVIII primers. HPRT served as internal control. Following PCR, bands were visualized using silver staining. The blots are representative of four experiments performed individually with similar results; (B) Graph represents hTERT or PKCδVIII densitometric units normalized to hypoxanthine guanine phosphoribosyl transferase (HPRT—a housekeeping gene used in RT-PCR) and is representative of mean ± SD in four experiments performed independently. Statistical analysis performed by two-tail Student’s t-test; ***P<0.001 highly significant for hTERT between days 0, 4, 8 of lean ADSC and obese ADSC; and lean ADSC and lean over-expressing PKCδVIII ADSC. hTERT, human telomerase reverse transcriptase; ADSC, adipose-derived stem cells; PKCδ, protein kinase C delta.

Figure 4

PKCδVIII over-expression increases telomerase. Subcutaneous lean ADSC control or over-expressing PKCδVIII were differentiated in vitro and whole cell lysates were collected on days 0, 4 and 8. (A) Western blot analysis was performed and immunoblotted using telomerase antibody or PKCδVIII-specific antibody. The blots are representative of three experiments performed individually with similar results; (B) Graph represents telomerase or PKCδVIII densitometric units normalized to β-actin and is representative of mean ± SD in four experiments performed independently. Statistical analysis performed by two-tail Student’s t-test; ***P<0.001 highly significant for telomerase between days 0, 4, 8 of lean ADSC and lean over-expressing PKCδVIII ADSC. ADSC, adipose-derived stem cells; PKCδ, protein kinase C delta.

Figure 5

PKCδVIII over-expression and senescence. (A) Lean ADSC and obese ADSC or (B) Subcutaneous lean ADSC or lean ADSC over-expressing PKCδVIII were stained for β-galactosidase to detect senescence. Cells were then visualized under 4× or 10× light microscopy using Nikon Eclipse microscope. The percent of cells staining positive was calculated by counting five areas of the same size from each field per sample using NIS elements advance research image tool software; (C,D) The graphs show average of β-gal positive cells. Statistical analysis performed by two-tail Student’s t-test; **P<0.01 significant for β-gal between lean ADSC and lean over-expressing PKCδVIII ADSC; and lean ADSC and obese ADSC. ADSC, adipose-derived stem cells; PKCδ, protein kinase C delta.