Influence of Tumor Necrosis Factor α, Parathyroid Hormone, and Vitamin D3 on Modulation of the RANKL2 Isoform: A Pilot Study

Abstract

RANKL exists as three isoforms: RANKL1, 2, and 3. RANKL1 and 3 were reported to be differently expressed upon treatment with some osteotropic factors, but RANKL2 expression could not be reliably determined. Here, we investigated through a mechanistic model, human 293 cells stably transfected with the RANKL2cDNA, the production and modulation of RANKL2 protein stability upon treatment with TNF-α, vitamin D3, and PTH. Data showed that TNF-a significantly increased (p<0.03) RANKL2 production and its half-life/stability (p<0.005). Vitamin D3 and PTH had no effect. This information will help to better define and differentiate the pathological mechanisms operating during osteolytic diseases.

Keywords: PTH; RANKL2 isoform; TNF-α; bone; cytokines and growth factors; osteoarthritis disease modification; preclinical research; vitamin D3.

Figure 1.

(A) Expression and (B) production of RANKL2 from the 293 cells and 293RANKL2 cells as determined by semiquantitative PCR and Western blot, respectively. (C) RANKL2 protein modulation in 293RANKL2 (n = 5) cells incubated with vitamin D₃ (50 nM), TNF-α (5 ng/mL), and PTH (100 nM). Cells were incubated for 72 hours in the presence of the factors, and RANKL2 protein levels were determined in the cell lysates using a specific ELISA. Data are expressed over the control, which was attributed a value of 1. Statistical analysis was assessed by the Student t test, and the P value is as indicated.

Figure 2.

RANKL2 protein stability/half-life modulation in 293RANKL2 (n = 3) following incubation with an increased time period with actinomycin D (5 µg/mL) and (A) vitamin D₃ (50 nM) or (B) TNF-α (5 ng/mL). RANKL2 protein levels were determined in the cell lysates using a specific ELISA. Data are expressed over the control, which was attributed a value of 1. Statistical analysis was assessed by the Student t test, and the P value is as indicated.