Figure 1. Osteolectin contains conserved integrin binding motifs and binds with high affinity to integrin α₁₁β₁.

(A, B) The human (A) and mouse (B) Osteolectin proteins contain RGD and LDT sequences. (C) Alignment of Osteolectin amino acid sequences shows that the RGD and LDT domains are evolutionarily conserved among bony vertebrates. (D, E) RNA-seq analysis of integrin α (D) and β (E) subunits in PDGFRα⁺CD45^-Ter119^-CD31^- bone marrow stromal cells from enzymatically dissociated adult bone marrow (n = 2 independent samples). These cells are uniformly positive for LepR expression (Zhou et al., 2014). (F) RNA-seq analysis of Itga1, Itga6, Itga11, and Itgav in PDGFRα⁺CD45^-Ter119^-CD31^- bone marrow stromal cells, VE-Cadherin⁺ bone marrow endothelial cells, and whole bone marrow cells (n = 2 independent samples per cell population). (G) Itga11 expression in cell populations from mouse bone marrow by qRT-PCR (n = 3 independent samples per cell population). The markers used for the isolation of each cell population are shown in Supplementary file 1. (H) In MC3T3-E1 preosteoblast cells expressing Flag-tagged Osteolectin, anti-Flag antibody co-immunoprecipitated endogenous integrin β1 and integrin α11 with Flag-tagged Osteolectin (results are representative of two independent experiments). (I) Recombinant human Osteolectin (rhOln) selectively bound to recombinant human integrin α₁₁β₁ and α₁₀β₁, but not to other integrins (n = 3 independent experiments). (J) Integrin α11β1 bound Osteolectin and recombinant human Pro-Collagen 1α (rhCol1A) with similar affinities, but not bovine serum albumin (BSA) (n = 3 independent experiments). (K) Osteolectin, but not Pro-Collagen 1α, promoted osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells (n = 3 independent experiments). (L) 200 nM RGDS peptide inhibited the binding of integrin α11β1 to recombinant human Osteolectin. (M) 100 μM RGDS peptide inhibited osteogenic differentiation by MC3T3-E1 cells and human bone marrow stromal cells in response to 30 ng/ml of recombinant human Osteolectin. All numerical data reflect mean ±standard deviation. Statistical significance was determined with one-way (G) or two-way ANOVAs with Dunnett’s multiple comparisons tests (K) or Tukey’s multiple comparisons tests (M).