Dipeptidyl Peptidase-4 and Adolescent Idiopathic Scoliosis: Expression in Osteoblasts

Abstract

It has been proposed that girls with adolescent idiopathic scoliosis (AIS) tend to have a taller stature and a lower body mass index. Energy homeostasis, that is known to affect bone growth, could contribute to these characteristics. In circulation, dipeptidyl peptidase-4 (DPP-4) inactivates glucagon-like peptide-1 (GLP-1), an incretin that promotes insulin secretion and sensitivity. Our objectives were to investigate DPP-4 status in plasma and in osteoblasts of AIS subjects and controls and to evaluate the regulatory role of metabolic effectors on DPP-4 expression. DPP-4 activity was assessed in plasma of 113 girls and 62 age-matched controls. Osteoblasts were isolated from bone specimens of AIS patients and controls. Human cells were incubated with glucose, insulin, GLP-1 and butyrate. Gene and protein expressions were evaluated by RT-qPCR and Western blot. Our results showed 14% inferior plasma DPP-4 activity in AIS patients when compared to healthy controls (P = 0.0357). Similarly, osteoblasts derived from AIS subjects had lower DPP-4 gene and protein expression than controls by 90.5% and 57.1% respectively (P < 0.009). DPP-4 expression was regulated in a different manner in osteoblasts isolated from AIS participants compared to controls. Our results suggest a role for incretins in AIS development and severity.

Figure 1

Plasma DPP-4 activity. DPP-4 activity was measured using the DPPIV/CD26 Enzo Life Science’s assay kit in plasma of 113 AIS girls and 62 age-matched controls. *P < 0.05 using two-tailed Student’s t-test. Data are presented as mean ± standard deviation.

Figure 2

DPP-4 gene and protein expression in osteoblasts isolated from controls and AIS patients. (a) DPP4 gene expression was measured by RT-qPCR with GAPDH as endogenous control (n = 7/group). Relative expression was analyzed with the 2^−ΔΔCT method. (b) DPP-4 protein expression was evaluated by Western blot (n = 3 controls and n = 6 AIS patients) and β-actin was used as endogenous control. **P < 0.01 using two-tailed Student’s t-test. Data are presented as mean ± standard deviation.

Figure 3

Impact of short-term treatments with glucose and insulin on DPP-4 expression. Cells were treated with glucose (5 mM), insulin (0.3 nM) and glucose + insulin (5 mM and 0.3 nM) for 2 hours. (a) DPP4 gene expression in osteoblasts of controls (n = 3) and AIS patients (n = 4–6) was measured by RT-qPCR with GAPDH as endogenous control. Relative expression was analyzed with the 2^−ΔΔCT method. (b) DPP-4 protein expression in osteoblasts of controls and AIS patients (n = 3/group) was measured by Western blot with β-actin as endogenous control. *P < 0.05 and **P < 0.01 vs. untreated controls; ^#P < 0.05 and ^##P < 0.01 vs. treated controls using one-way ANOVA followed by Tukey’s post-hoc tests. Data are presented as mean ± standard deviation.

Figure 4

Impact of long-term treatments with glucose and insulin on DPP-4 expression. Cells were treated with glucose (5 mM), insulin (0.3 nM) and glucose + insulin (5 mM and 0.3 nM) for 24 hours. (a) DPP4 gene expression in osteoblasts of controls (n = 3) and AIS patients (n = 4–6) was measured by RT-qPCR with GAPDH as endogenous control. Relative expression was analyzed with the 2^−ΔΔCT method. (b) DPP-4 protein expression in osteoblasts of controls and AIS patients (n = 3/group) was measured by Western blot with β-actin as endogenous control. *P < 0.05 and **P < 0.01 vs. untreated controls; ^##P < 0.01 and ^###P < 0.001 vs. treated controls using one-way ANOVA followed by Tukey’s post-hoc tests. Data are presented as mean ± standard deviation.

Figure 5

Impact of long-term treatment with GLP-1 on DPP-4 expression. Cells were treated with GLP-1 (10 mM) for 24 hours. (a) DPP4 gene expression was measured in osteoblasts of controls (n = 3) and AIS patients (n = 6) by RT-qPCR with GAPDH as endogenous control. Relative expression was analyzed with the 2^−ΔΔCT method. (b) DPP-4 protein expression in osteoblasts of controls and AIS patients (n = 3/group) was measured by Western blot with β-actin as endogenous control. **P < 0.01 vs. untreated controls; ^###P < 0.001 vs. treated controls using one-way ANOVA followed by Tukey’s post-hoc tests. Data are presented as mean ± standard deviation.

Figure 6

STAT1 gene and protein expression in osteoblasts isolated from control and AIS patients. (a) STAT1 gene expression was measured in osteoblasts of controls (n = 7) and AIS patients (n = 13) by RT-qPCR with GAPDH as endogenous control. Relative expression was analyzed with the 2^−ΔΔCT method. (b) STAT-1 protein expression was evaluated by Western blot (n = 3/group) and β-actin was used as endogenous control. *P < 0.05 using two-tailed Student’s t-test. Data are presented as mean ± standard deviation.