Overexpression of Neurogenin 1 Negatively Regulates Osteoclast and Osteoblast Differentiation

Abstract

Neurogenin 1 (Ngn1) belongs to the basic helix-loop-helix (bHLH) transcription factor family and plays important roles in specifying neuronal differentiation. The present study aimed to determine whether forced Ngn1 expression contributes to bone homeostasis. Ngn1 inhibited the p300/CREB-binding protein-associated factor (PCAF)-induced acetylation of nuclear factor of activated T cells 1 (NFATc1) and runt-related transcription factor 2 (Runx2) through binding to PCAF, which led to the inhibition of osteoclast and osteoblast differentiation, respectively. In addition, Ngn1 overexpression inhibited the TNF-α- and IL-17A-mediated enhancement of osteoclast differentiation and IL-17A-induced osteoblast differentiation. These findings indicate that Ngn1 can serve as a novel therapeutic agent for treating ankylosing spondylitis with abnormally increased bone formation and resorption.

Keywords: Neurogenin 1; PCAF; ankylosing spondylitis; osteoblast; osteoclast.

Figure 1

The overexpression of Neurogenin 1 (Ngn1) inhibited the receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. (a,b) Bone marrow-derived macrophages (BMMs) overexpressing Ngn1 were generated via retroviral infection and cultured with a macrophage colony stimulating factor (M-CSF) and RANKL for 3 days. (a) Tartrate-resistant acid phosphatase (TRAP)-stained cells (left panel) and the number of TRAP-positive multinucleated cells (right panel), scale bar: 200 µm. (b) Relative expression of the indicated genes quantified using quantitative reverse-transcription PCR (qRT-PCR). # p < 0.05, * p < 0.01, ** p < 0.001 vs. control.

Figure 2

The overexpression of Ngn1 strongly inhibited osteoblast differentiation. (a–c) Osteoblast precursor cells overexpressing Ngn1 were generated via retroviral infection and incubated in an osteogenic medium (OGM). (a) Levels of alkaline phosphatase (ALP) in cells cultured for 3 days. (b) The cells were cultured for 6 days, stained with alizarin red (left panel), and those positive for alizarin red were quantified (right panel). (c) Relative expression of the indicated genes quantified via quantitative reverse-transcription PCR (qRT-PCR). # p < 0.05, * p < 0.01, ** p < 0.001 vs. control.

Figure 3

Ngn1 regulated the p300/CREB-binding protein-associated factor (PCAF)-mediated transcriptional activities of the nuclear factor of activated T cells 1 (NFATc1) and runt-related transcription factor 2 (Runx2). (a) The 293T cells were co-transfected with Flag-PCAF and Flag-Ngn1 and subjected to immunoprecipitation with an IgG or anti-PCAF antibody, followed by Western blot analysis with an anti-Flag antibody. (b) The 293T cells were co-transfected with HA-NFATc1, Flag-PCAF, or Flag-Ngn1, as indicated. Cell lysates were subjected to immunoprecipitation with an anti-acetyl-lysine antibody, followed by Western blot analysis of the indicated antibodies. (c) The 293T cells were co-transfected with Myc-Runx2, Flag-PCAF, or Flag-Ngn1, as indicated. Cell lysates were subjected to immunoprecipitation with an anti-acetyl-lysine antibody, followed by Western blot analysis with the indicated antibodies. (d) The 293T cells were co-transfected with the indicated plasmids along with an Acp5 or Oscar promoter luciferase reporter. Cell lysates were subjected to the luciferase assay. (e) The 293T cells were co-transfected with the indicated plasmids along with an Alpl, Bglap, or OSE promoter luciferase reporter. Cell lysates were subjected to the luciferase assay. # p < 0.05, * p < 0.01, ** p < 0.001 vs. control.

Figure 4

The overexpression of NFATc1 or Runx2 partially rescued the inhibitory effects of Ngn1 on osteoclastogenesis or osteoblastogenesis, respectively. (a) BMMs overexpressing Ngn1 or Ngn1 and Ca-NFATc1 were generated via retroviral infection and subsequently cultured with M-CSF and RANKL for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive multinucleated cells (right panel), scale bar: 200 µm. (b,c) Osteoblast precursor cells overexpressing Ngn1 or Ngn1 and Runx2 were generated via retroviral infection and subsequently cultured with OGM. (b) Levels of ALP in cells cultured for 3 days. (c) Cells were cultured for 6 days, stained with alizarin red (left panel), and those positive for alizarin red were quantified (right panel). # p < 0.05, * p < 0.01, ** p < 0.001 vs. control.

Figure 5

The overexpression of Ngn1 inhibited the osteoclastogenesis and osteoblastogenesis mediated by inflammatory cytokines. (a) BMMs overexpressing Ngn1 were generated via retroviral infection and subsequently incubated with M-CSF, RANKL, and TNF-α for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive multinucleated cells (right panel), scale bar: 200 µm. (b) BMMs overexpressing Ngn1 were generated via retroviral infection and subsequently incubated with M-CSF, RANKL, and IL-17A for 3 days. TRAP-stained cells (left panel) and the number of TRAP-positive multinucleated cells (right panel), scale bar: 200 µm. (c,d) Osteoblast precursor cells overexpressing Ngn1 were generated via retroviral infection and subsequently cultured with ascorbic acid, β-glycerophosphate, and IL-17A. (c) Levels of ALP in cells cultured for 3 days. (d) Cells were cultured for 6 days, stained with alizarin red (left panel), and those positive for alizarin red were quantified (right panel). * p < 0.01, ** p < 0.001 vs. control.