Background: The extracellular matrix (ECM) makes a physical framework for cells in bone which initiates biochemical signaling required for key cellular events such as adhesion, differentiation, migration, proliferation and survival. Hypoxia is a critical regulator of ECM remodeling. In our previous observations, we found that Mucin 1 (MUC1) could counter the effect of hypoxia in human osteoblasts.

Objective: We wanted to assess the MUC1-mediated effect of hypoxia on proteomic composition of human osteoblast-derived extracellular matrix.

Methods: Human bone marrow-derived mesenchymal stromal cells (MSCs) were osteogenically differentiated into osteoblasts under normoxia (20% O₂) or hypoxia (2% O₂) with or without 5 μM GO-201 (MUC1 inhibitor). On day 11, osteoblasts were devitalized using freeze-thawing cycles and DNase treatment. Proteomic composition of ECMs was analyzed using LCMS with a label-free quantification (LFQ) method.

Results: Proteomic analysis revealed 1765 proteins to be expressed in all four conditions. There were 267 and 165 proteins uniquely expressed under hypoxia and normoxia, respectively. Hypoxia up- and downregulated expression of 71 and 22 proteins by more than two fold, respectively compared to normoxia. The number of proteins with altered expression was lowest (11 upregulated and 12 downregulated) for the hypoxia versus hypoxia+GO-201 comparison. The gene ontology term “cell-cell adherens junction” was highly enriched under hypoxia (hypoxia versus normoxia comparison) as well as under hypoxia+GO-201 (hypoxia versus hypoxia+GO-201 comparison). Ingenuity pathway analysis (IPA) showed that hypoxia is associated with proteins related to activation of apoptosis, whereas additional MUC1 inhibition caused the opposite effect.

Conclusion: IPA analysis shows that osteoblast ECM generated under hypoxia is involved in differentiation inhibition of osteoblasts and activation of apoptosis, both of which are opposed by MUC1 inhibition under hypoxia. MUC1 inhibition under hypoxia may therefore create a favorable ECM composition to promote osteogenesis.