The novel pro-osteogenic activity of NUCB2(1-83.)

Abstract

NUCB2¹⁻⁸³ has been recently reported as an anorexigenic and anti-hyperglycemic peptide. Here we report that NUCB2¹⁻⁸³ promotes osteogenesis. It was found after two months of once-a-day intravenous injection of NUCB2¹⁻⁸³, bone mineral density of femora and lumbar vertebrae were increased in ovariectomized rats. NUCB2¹⁻⁸³ also increased the alkaline phosphatase activity and promoted mineralization in mouse MC3T3-E1 preosteoblastic cell line. When either both Arg⁶⁰ and Arg⁶³ or Ser⁷² were mutated to Ala, the pro-osteogenic activity was completely lost, indicating that these residues are structurally important for its biological function. Furthermore, it encumbered osteoclastic differentiation of RAW 264.7 macrophage. It also excluded any possibility of the effect caused by contaminants or experimental faults, and demonstrated that the pro-osteogenic activity observed was a specific effect of NUCB2¹⁻⁸³ itself. These findings warranted that further studies on NUCB2¹⁻⁸³ would be valuable for the treatment of bone metabolic diseases especially for osteoporosis.

Figure 1. BMD of femora and lumbar vertebrae in the OVX model and experiment groups.

(A) BMD in OVX and sham-operated groups. Three-month-old virgin female Sprague-Dawley rats were ovariectomized or sham-operated. They were euthanized at 16 weeks after surgery, and their femora and lumbar vertebrae were dissected free of soft tissues and stored in 75% ethanol for the BMD assay. (B) BMD in different experimental groups after 60-day continuous administration. The ovariectomized rats were divided into two groups and intravenously injected with saline or NUCB2^1–83 (50 nmol) once a day. After 60 days, their femora and lumbar vertebrae were collected for the BMD assay. Data represented the mean±SEM(*P<0.05). Number of bone samples used is showed within the parentheses.

Figure 2. Dose-responsive effects of NUCB2^1–83 on the ALP activity of MC3T3-E1.

MC3T3-E1 cells were treated with test samples for six days in the presence of rhBMP-2 (1 µg/mL). Data represented the mean±SEM. Number of samples used is showed within the parentheses. Statistical analysis according to one-way ANOVA was followed by Dunnett's test (**P<0.01).

Figure 3. The effect of NUCB2^1–83 and its mutants on the ALP activity of MC3T3-E1.

MC3T3-E1 cells were treated with test samples for six days in the presence of rhBMP-2 (1 µg/mL). The concentrations of NUCB2^1–83 and its mutants were 10 nmol/mL. Data represented the mean±SEM. Number of samples used is showed within the parentheses. Statistical analysis according to one-way ANOVA was followed by Dunnett's test for BMP-2 vs. test samples (**P<0.01, ***P<0.001).

Figure 4. The effect of NUCB2^1–83 on mineralization measured by Alizarin Red staining.

MC3T3-E1 cells were cultured for 15 days with or without NUCB2^1–83 and the mineralized matrices were stained by Alizarin-red staining method (A) NUCB2^1–83 (B) control group just with conditional medium (C) The mineralized nodules were observed by microscope (magnification 100×).

Figure 5. The effect of NUCB2^1–83 on Mineralization quantified by measuring calcium levels.

MC3T3-E1 cells were cultured for 15 days with or without NUCB2^1–83 and lysates were collected for calcium measurement using the commercial calcium kit. Data represented the mean±SEM(**P<0.01). Number of samples used is showed within the parentheses.

Figure 6. The effect of NUCB2^1–83 on osteoclastogenesis of RAW 264.7 measured by TRAP staining.

RAW 264.7 cells were cultured in the presence of RANKL (50 ng/mL) for 5 days with or without NUCB2^1–83 and stained for TRAP using leukocyte acid phosphatase kit. TRAP-positive multinucleated cells were visualized under light microscopy. (A,B) control group just with α-MEM (C,D) RANKL (E,F) RANKL and NUCB2^1–83. B, D, F were taken at 1000× magnification.

Figure 7. The effect of NUCB2^1–83 on osteoclast differentiation quantified by measuring TRAP activity.

RAW 264.7 cells were cultured in the presence of RANKL (50 ng/mL) for 5 days with or without NUCB2^1–83. The cells were fixed and incubated in 10 mM citrate buffer (pH 4.6) containing 10 mM sodium tratrate and 5 mM p-nitrophenylphosphate for 1 h followed by transferring into new well plates containing an equal volume of 0.1 N NaOH. TRAP activity was measured at λ = 405 nm and expressed as percent of that of untreated control. Data represented the mean±SEM(*P<0.05). Number of samples used is showed within the parentheses.

Figure 8. Expression of NUCB2^1–83 mRNA in MC3T3-E1 cells and differentiated RAW 264.7 cells.

The precise quantitative data were obtained by using Real-Time Quantitative PCR. The results were normalized to beta-actin, which served as a control to verify the amount of samples. Data represented the mean±SEM. Number of samples used is showed within the parentheses.