Inhibition of hypoxia-induced Mucin 1 alters the proteomic composition of human osteoblast-produced extracellular matrix, leading to reduced osteogenic and angiogenic potential

Abstract

The bone microenvironment is one of the most hypoxic regions of the human body and in experimental models; hypoxia inhibits osteogenic differentiation of mesenchymal stromal cells (MSCs). Our previous work revealed that Mucin 1 (MUC1) was dynamically expressed during osteogenic differentiation of human MSCs and upregulated by hypoxia. Upon stimulation, its C-terminus (MUC1-CT) is proteolytically cleaved, translocases to the nucleus, and binds to promoters of target genes. Therefore, we assessed the MUC1-mediated effect of hypoxia on the proteomic composition of human osteoblast-derived extracellular matrices (ECMs) and characterized their osteogenic and angiogenic potentials in the produced ECMs. We generated ECMs from osteogenically differentiated human MSC cultured in vitro under 20% or 2% oxygen with or without GO-201, a MUC1-CT inhibitor. Hypoxia upregulated MUC1, vascular endothelial growth factor, and connective tissue growth factor independent of MUC1 inhibition, whereas GO-201 stabilized hypoxia-inducible factor 1-alpha. Hypoxia and/or MUC1-CT inhibition reduced osteogenic differentiation of human MSC by AMP-activated protein kinase/mTORC1/S6K pathway and dampened their matrix mineralization. Hypoxia modulated ECMs by transforming growth factor-beta/Smad and phosphorylation of NFκB and upregulated COL1A1, COL5A1, and COL5A3. The ECMs of hypoxic osteoblasts reduced MSC proliferation and accelerated their osteogenic differentiation, whereas MUC1-CT-inhibited ECMs counteracted these effects. In addition, ECMs generated under MUC1-CT inhibition reduced the angiogenic potential independent of oxygen concentration. We claim here that MUC1 is critical for hypoxia-mediated changes during osteoblastogenesis, which not only alters the proteomic landscape of the ECM but thereby also modulates its osteogenic and angiogenic potentials.

Keywords: angiogenesis; extracellular matrix; human osteoblasts; hypoxia; mucin-1.

Figure 1

Effect of hypoxia and MUC1‐CT inhibition on HIF1α, MUC1, and VEGF expression. (a) HIF1α protein expression on Days 1 and 6 of osteogenic differentiation of human MSC under 20% or 2% oxygen with or without GO‐201. (b) Gene expression of MUC1 on the Days 1, 6, 11, and 19. (c) Protein expression of MUC1 on Day 11. (d) Gene expression of VEGF on Days 1, 6, 11, and 19. Ribosomal phosphoprotein 36B4 was used as a housekeeping gene and vinculin or α‐tubulin was used to correct for protein expression. Results are representative for multiple independent experiments (*p < 0.05; **p < 0.005; ***p < 0.001). Bars represent averages ± SD. HIF1α, hypoxia‐inducible factors 1α; MUC1, Mucin 1; VEGF, vascular endothelial growth factor

Figure 2

Effect of hypoxia and MUC1‐CT inhibition on osteogenic differentiation of MSCs. (a) Mineralization of extracellular matrix (ECM) on Days 19 and 24 of osteogenic differentiation of MSC under 20% or 2% oxygen with or without GO‐201. (b) Mineralization of ECM on Day 17 of osteogenic differentiation of MSC under 20% or 2% oxygen with scrambled (Scr) or short hairpin RNAs against MUC1 (Sh1 and Sh2). (c) Alkaline phosphatase (ALP) activity measured in cell lysates on Day 11 of osteogenic differentiation of human MSC under 20% or 2% oxygen with or without GO‐201 (*p < 0.05; ***p < 0.001). Bars represent averages ± SD. MSC, mesenchymal stromal cell; MUC1, Mucin 1

Figure 3

Effect of hypoxia and MUC1‐CT inhibition on the proteomic landscape of ECM of osteogenic differentiation of MSCs. (a) Pathway enrichment analysis of proteins detected in hypoxic ECM versus normoxic ECM; proteins either uniquely present or up‐or downregulated (≥2‐fold) between the two groups were included for the analysis. Activated pathways are shown in orange and deactivated pathways are shown in blue. (b) Pathway enrichment analysis of proteins detected in normoxic‐GO‐201 ECM versus normoxic ECM; proteins either uniquely present or up‐or downregulated (≥2‐fold) between the two groups were included for the analysis. Activated pathways are shown in orange and deactivated pathways are shown in blue. (c) Pathway enrichment analysis of proteins detected in hypoxic‐GO‐201 ECM versus hypoxic ECM; proteins either uniquely present or up‐or downregulated (≥2‐fold) between the two groups were included for the analysis. Activated pathways are shown in orange and deactivated pathways are shown in blue. (d) Pathway enrichment analysis of proteins detected in hypoxic‐GO‐201 ECM versus normoxic‐GO‐201 ECM; proteins either uniquely present or up‐or downregulated (≥2‐fold) between the two groups were included for the analysis. Activated pathways are shown in orange and deactivated pathways are shown in blue. (e) Heat map representing the abundance (LFQ values) of all the collagen proteins detected in the four groups of ECM samples combined. Red: high abundance, blue: low abundance, grey: not detected. (f) Gene expression of COL1A1, COL5A1, and COL5A3 on Day 11 of osteogenic differentiation of human MSC under 20% or 2% oxygen with or without GO‐201. Ribosomal phosphoprotein 36B4 was used as a housekeeping gene (*p < 0.05; **p < 0.005; ***p < 0.001). Bars represent averages ± SD. ECM, extracellular matrix; MSC, mesenchymal stromal cell; MUC1, Mucin 1

Figure 4

Effect of hypoxia and MUC1‐CT inhibition on AMPK/mTORC1/S6K pathway in osteogenic differentiation of MSCs. (a) Immunoblots of protein expression on Day 1 of osteogenic differentiation of MSC cultured under 20% or 2% oxygen with or without GO‐201. (b) Quantification of phosphorylated over total protein ratio's relative to β‐actin (*p < 0.05). Bars represent averages ± SD. AMPK, AMP‐activated protein kinase; MSC, mesenchymal stromal cell; MUC1, Mucin 1

Figure 5

Effect of hypoxia and MUC1‐CT inhibition on IKK/NFκB pathway in osteogenic differentiation of MSCs. (a, b) Gene expression of TGFβ1 (left panel) and TGFβ2 (right panel). (c–f) Gene expression of SMAD2, SMAD4, SMAD3, and SMAD7. Ribosomal phosphoprotein 36B4 was used as a housekeeping gene. (g) Immunoblots of protein expression on Day 1 (left panels) and Day 6 (right panels) of osteogenic differentiation of MSC cultured under 20% or 2% oxygen with or without GO‐201. (h) Quantification of proteins relative to β‐tubulin (*p < 0.05; **p < 0.005; ***p < 0.001). Bars represent averages ± SD. MSC, mesenchymal stromal cell; MUC1, Mucin 1; NFκB, nuclear factor‐κB; TGFβ, transforming growth factor‐beta

Figure 6

Effect of ECMs generated from normoxic or hypoxic osteoblasts with or without GO‐201 on osteogenic differentiation of MSC. (a) Metabolic activity of MSC cultured on plastic or different ECMs for 6, 24, and 48 h. (b) Percentage of Ki‐67 positive MSC cultured for 3 days over plastic or different ECMs counted by flow cytometry. (c) Alkaline phosphatase (ALP) activity of osteogenically differentiating MSC on Days 6, 11, and 19 cultured on plastic or different ECMs. (d) Mineralization of extracellular matrix (ECM) of osteogenically differentiating MSC on Days 14, 17, and 21 cultured on plastic or different ECMs (*p < 0.05; **p < 0.005; ***p < 0.001). Bars represent averages ± SD. ECM, extracellular matrix; MSC, mesenchymal stromal cell

Figure 7

Effect of ECMs generated from normoxic or hypoxic osteoblasts with or without GO‐201 on the behavior of HUVECs. (a) Gene expression of Ki‐67 in HUVECs cultured for 5 days on plastic or ECMs generated from osteogenically differentiated human MSC under 20% or 2% oxygen with or without GO‐201. (b) Percentage of Ki‐67 positive HUVECs cultured for 3 days over plastic or different ECMs counted by flow cytometry. (c–f) Gene expression at Day 3 of TIE‐1, TIE‐2, ANG‐1, and ANG‐2. (g) The ratio of mRNA expression of BAX/BCL2. Ribosomal phosphoprotein 36B4was used as housekeeping gene (*p < 0.05; **p < 0.005; ***p < 0.001). Bars represent averages ± SD. ECM, extracellular matrix; HUVEC, human umbilical vein endothelial cell; mRNA, messenger RNA

Figure 8

Schematic model of hypoxia‐induced Mucin 1 regulating the osteogenic differentiation of human mesenchymal stromal cells and properties of their extracellular matrix. MUC1 is moderately expressed in osteoblasts under 20% oxygen (normoxia) (blue arrow in the yellow panel). Inhibition of MUC1 by GO‐201 (red symbol) under normoxic conditions reduced ECM mineralization, activated NFkB pathway, reduced osteogenic, and angiogenic potentials of ECMs in osteoblasts (in the left yellow panel). Hypoxia upregulated MUC1 and reduced mineralization of ECM in osteoblasts (blue arrow in the white panel). Hypoxic ECM had higher osteogenic potential compared to normoxic ECM that was reversed by MUC1 inhibition under hypoxia (white panel). ECM, extracellular matrix; MUC1, Mucin 1; NFκB, nuclear factor‐κB