Progression of human bone marrow stromal cells into both osteogenic and adipogenic lineages is differentially regulated by structural conformation of collagen I matrix via distinct signaling pathways

Abstract

Adult human bone marrow stromal cells (BMSCs) containing or consisting of mesenchymal stem cells (MSCs) are an important source in tissue homeostasis and repair. Although many processes involved in their differentiation into diverse lineages have been deciphered, substantial inroads remain to be gained to synthesize a complete regulatory picture. The present study suggests that structural conformation of extracellular collagen I, the major organic matrix component in musculoskeletal tissues, plays, along with differentiation stimuli, a decisive role in the selection of differentiation lineage. It introduces a novel concept which proposes that structural transition of collagen I matrix regulates cell differentiation through distinct signaling pathways specific for the structural state of the matrix. Thus, on native collagen I matrix inefficient adipogenesis is p38-independent, whereas on its denatured counterpart, an efficient adipogenesis is primarily regulated by p38 kinase. Inversely, osteogenic differentiation occurs efficiently on native, but not on denatured collagen I matrix, with a low commencement threshold on the former and a substantially higher one on the latter. Osteogenesis on collagen I matrices in both structural conformations is fully dependent on ERK. However, whereas on native collagen I matrix osteogenic differentiation is Hsp90-dependent, on denatured collagen I matrix it is Hsp90-independent. The matrix conformation-mediated regulation appears to be one of the mechanisms determining differentiation lineage of BMSCs. It allows a novel interpretation of the bone remodeling cycle, explains the marked physiological aging-related adipogenic shift in musculoskeletal tissues, and can be a principal contributor to adipogenic shift seen in a number of clinical disorders.

Figure 1.. Adipogenic differentiation of bone marrow stromal cells is substantially more efficient on denatured than on native collagen I matrix.

DC: denatured collagen I matrix; NC: native collagen I matrix; AD: adipogenic stimulants. FABP4: fatty acid-binding protein-4; LPL: lipoprotein lipase; GLUT4: facilitative glucose transporter-4. Photomicrographs insert: scale bar = 500μm. Values obtained with AD-treated samples were significantly different in comparison to untreated controls, Δ= p<0.05.

Figure 2.. Adipogenic differentiation of bone marrow stromal cells is p38-independent and suppressed by ERK on native collagen I matrix but is regulated primarily by p38 on its denatured counterpart.

C: untreated controls; AD: adipogenic stimuli; SB203580, PD98059: inhibitors of p38 and ERK, respectively. NC: native collagen I matrix; DC: denatured collagen I matrix. (+AD) samples: significantly different in comparison to untreated controls; p<0.05. [Δ= p<0.05]: significantly different in comparison to (NC+AD). [α= p<0.05]: significantly different in comparison to (DC+AD); [α= p>0.05]: statistically similar to (NC+AD). Photomicrographs inset: scale bar = 500μm.

Figure 3.. Osteogenic differentiation of bone marrow stromal cells is substantially more efficient on native than on denatured collagen I matrix.

DC: denatured collagen I matrix; NC: native collagen I matrix; OS: osteogenic stimulation. BSP: bone sialoprotein; MGP: matrix GLA protein; Runx2, runt-related transcription factor 2. Values for BSP, MGP, and Runx2 expression were determined after 14 days of OS treatment; calcium deposition was measured after 21 days. Values obtained with OS-treated samples were significantly different in comparison to untreated controls, *= p<0.05.

Figure 4.. Osteogenic differentiation of bone marrow stromal cells is regulated mainly by ERK and suppressed by p38 on both native and denatured collagen I matrices.

C: untreated controls; OS: osteogenic stimuli; PD98059, SB203580: inhibitors of ERK and p38, respectively. NC: native collagen I matrix; DC: denatured collagen I matrix. (+OS) samples: significantly different in comparison to respective untreated controls, p<0.05. [*= p<0.05]: significantly different in comparison to (DC+OS). [μ= p<0.05]: significantly different in comparison to respective OS-treated cells on NC and DC. [δ = p>0.05]: statistically similar in comparison to untreated controls.

Figure 5.. Osteogenic differentiation of bone marrow stromal cells is Hsp90-dependent on native collagen I matrix but Hsp90-independent on its denatured counterpart.

C: untreated controls; OS: osteogenic stimuli; 17-AAG: 17-Allylamino-17-demethoxygeldanamycin, inhibitor of Hsp90. NC: native collagen I matrix; DC: denatured collagen I matrix. (+OS) samples: significantly different in comparison to untreated controls, p<0.05. [*= p<0.05]: significantly different in comparison to (DC+OS). [δ= p<0.05]: significantly different in comparison to (NC+OS); [δ= p<0.05]: statistically similar to untreated controls. [Δ= p>0.05]: statistically similar in comparison to (DC+OS).

Figure 6.. Inhibition of Hsp90 substantially elevates the extent of adipogenesis in bone marrow stromal cells on native collagen I matrix.

C: untreated controls; AD: adipogenic stimuli; 17-AAG: 17-Allylamino-17-demethoxygeldanamycin, inhibitor of Hsp90. NC: native collagen I matrix; DC: denatured collagen I matrix. +AD samples: significantly different in comparison to untreated controls; p<0.05. [α= p<0.05]: significantly different in comparison to cells cultured on (NC+AD). Photomicrographs inset: scale bar = 500μm.

Figure 7.. Osteogenic differentiation of bone marrow stromal cells occurs at much lower inducer concentration on native collagen I matrix then on its denatured counterpart.

Osteogenesis of mesenchymal stem cells on native and denatured collagen I matrices as function of dexamethasone concentration. BSP: bone sialoprotein; NC: native collagen I matrix; DC: denatured collagen I matrix.

Figure 8.. Bone marrow stromal cells on both native and denatured collagen I matrices do not commit irreversibly to specific osteogenic pathway.

Cells that underwent osteogenic differentiation on native or denatured collagen I matrices for five days were replated onto either identical or structurally opposite collagen I matrices in the presence of osteogenic stimuli for additional fourteen days. DC: denatured collagen I matrix; NC: native collagen I matrix; OS: osteogenic stimulation. BSP: bone sialoprotein; MGP: matrix GLA protein; Runx2, runt-related transcription factor 2. [DC-DC +OS]: cells transferred from DC to DC; [DC-NC +OS]: cells transferred from DC to NC; [NC-NC +OS]: cells transferred from NC to NC; [NC-DC +OS]: cells transferred from NC to DC. All experimental groups treated with OS significantly different in comparison to respective untreated controls, p<0.05. [*= p>0.05]: statistically similar to [NC-NC+OS]; [Δ= p>0.05]: statistically similar to [DC-DC +OS].

Figure 9.. Role of conformational transition of collagen I matrix in bone remodeling.

Upper circle: normal remodeling cycle. Lower circle and side panel: possible role of irregularities in conformational transition of collagen I matrix in some bone-associated pathologies.