PMC

Altering RhoA activity in MC3T3-E1 cells. (A) MC3T3-E1 cells were adenovirally transduced with vectors encoding for GFP, V14-RhoA, or N19-RhoA or treated with 0.5 μg/ml C3 toxin and cultured on tissue culture polystyrene. After 2 days, active RhoA levels were measured by ELISA. Data represent mean ± SD (N = 3; ^a p < 0.001 relative to control cells). (B) Representative micrographs at 1 and 14 days (phase contrast: columns 1 and 3; fluorescence: columns 2 and 4) of MC3T3-E1 cells expressing V14-RhoA, seeded at confluence on compliant hydrogels and rigid tissue culture polystyrene (TCPS) control (scale bar, 250 μm).

Matrix rigidity regulates RUNX2 expression in a RhoA-, ROCK-, and MAPK-dependent fashion. RUNX2 protein expression levels were analyzed by immunostaining MC3T3-E1 cells seeded at confluency on compliant hydrogels and rigid polystyrene (TCPS) substrate. Shown are representative immunofluorescent micrographs of MC3T3-E1 cells in which RUNX2 is shown in red, and nuclei are shown in blue (scale bars, 50 μm). (A) After 1 day, RUNX2 expression was evident only in cells cultured on rigid polystyrene (TCPS) but not in cells cultured on hydrogels, and was dependent on level of RhoA activity. (B) MC3T3-E1 cells expressing V14-RhoA showed elevated RUNX2 levels on stiffer matrices (stiff hydrogel and TCPS), whereas cells treated with PD98059, Y27632, or C3 had diminished RUNX2 staining. (C) RUNX2 expression correlated with ECM rigidity in wildtype and V14-RhoA expressing cells, increasing with increasing ECM rigidity. Almost no RUNX2 staining was evident in MC3T3-E1 cells treated with 50 μM PD98059, 10 μM Y27632, or 0.5 μg/ml C3 toxin.

RhoA, ROCK, and MAPK inhibition decreases ECM compliance–dependent ALP activity and diminishes matrix mineralization. (A and B) Wildtype and V14-RhoA expressing MC3T3-E1 cells were cultured on soft and stiff hydrogels, and control polystyrene (TCPS) either in the absence or presence of 50 μM PD98059, 10 μM Y27632, or 0.5 μg/ml C3 toxin. Cellular ALP activity (normalized by protein concentration) was measured after 1 (A) and 14 (B) days in culture. Data represent mean ± SD (N ≥ 3; ^a p < 0.05 relative to control wildtype cells; ^b p < 0.001 vs. control wildtype and V14-RhoA expressing cells). (C) MC3T3-E1 cells (wildtype and those adenovirally transduced with vectors encoding for GFP and V14-RhoA) were seeded at confluency in 12-well culture plates and cultured for 14 days either in the absence or presence of 50 μM PD98059, 10 μM Y27632, or 0.5 μg/ml C3 toxin as indicated. Shown are representative images at day 14 of MC3T3-E1 cells fixed and stained using the von Kossa technique for mineralized matrix (dark areas).

Inhibition of RhoA or ROCK decreases p44/42 MAPK (ERK1/2) phosphorylation. The levels of phosphorylated p44/42 MAPK (pMAPK) in wildtype and V14-RhoA expressing MC3T3-E1 cells, as a function of substrate stiffness, were detected using standard immunoblotting techniques at day 1 (A) and day 14 (C). β-actin was used as an internal loading control. (So, soft hydrogels; St, stiff hydrogels; PS, control TCPS substrates). Indicated groups were treated with 50 μM PD98059, 10 μM Y27632, or 0.5 μg/ml C3 toxin. (B and D) Quantification was performed using scanning densitometry. The levels of phosphorylated MAPK were normalized to levels of β-actin for each condition at both time points, and the ratios were compared with that of control cells on the soft hydrogel at day 1 (arbitrarily assigned a value of 1). Data represent mean ± SD (N ≥ 3; ^a p < 0.05, ^b p < 0.01, and ^c p < 0.001 relative to levels on soft hydrogels; ^d p < 0.01 relative to wildtype control and V14-RhoA expressing cells).

Model for regulation of osteogenic differentiation of MC3T3-E1 cells by substrate rigidity. In this model, changes in substrate compliance are transduced by integrins (such as α₂β₁, which binds to type I collagen) that cluster in plane of the plasma membrane, in turn stimulating downstream protein tyrosine kinases such as FAK to catalyze the activations of RhoA. On stiffer matrices, FAK activation is enhanced, leading to higher RhoA activity, thereby promoting increased cellular contractility through ROCK. Cross-talk between RhoA-ROCK and the ERK-MAPK pathway stimulates phosphorylation of ERK 1/2 (p44/42 MAPK) through MEK (MAPK kinase), which regulates the activity of the osteogenic transcription factor RUNX2 to control the expression of osteogenic genes (e.g., OCN and BSP) and ultimately drive differentiation toward a mature osteoblastic phenotype. On softer matrices in the presence of identical soluble cues, however, the diminished activity of FAK, RhoA, and ERK1/2 reduces the levels of RUNX2, inhibiting or at least delaying the subsequent steps of osteogenesis.

Mechanical and cell adhesive properties of poly(ethylene glycol) hydrogels are independently tunable. (A) Chemical structure of PEGDA molecule with acrylated end groups responsible for cross-linking. (B) Macroscopic images of a PEG hydrogel (1 in diameter) composed of 10 total polymer weight percent and a 50:50 ratio of PEGDA:PEG (arrow). (C) Complex shear moduli of PEG gels were assessed as a function of strain amplitude (strain sweep) or frequency (frequency sweep). Data represent mean ± SD (N = 5). (D) Representative phase contrast images of MC3T3-E1 cells cultured for 12 h on nonfunctionalized (left) and type I collagen functionalized (right) PEG hydrogels are shown (scale bar, 250 μm). (E) Shown are representative immunofluorescent micrographs of PEG hydrogels, composed of either 10 total polymer weight percent and a 50:50 ratio of PEGDA:PEG (top, denoted as soft hydrogel) or 20 total polymer weight percent containing 100% PEGDA (bottom, denoted as stiff hydrogel), coupled with the two different theoretical collagen densities (left: 5 μg/cm²; right: 50 μg/cm²). Scale bar represents 250 μm. (F) Quantification of such images showed a uniform fluorescent intensity for a given concentration irrespective of the substrate stiffness/chemistry. Data represent mean ± SD (N = 4; ^a p < 0.01 relative to 5 μg/cm² theoretical collagen density intensity levels).

Altered bone sialoprotein (BSP) and osteocalcin (OCN) gene expression levels in MAPK-, ROCK-, and RhoA-inhibited MC3T3-E1 cells. BSP, OCN, and type I collagen (α1 chain) gene expression levels in wildtype and V14-RhoA expressing MC3T3-E1 cells cultured on the three substrates were assessed by RT-PCR. GAPDH mRNA expression served as an internal control. Representative images of ethidium bromide–stained agarose gels at days 1 (A) and 14 (D) are shown (top panel: BSP, second panel: OCN, third panel: type-I collagen, bottom panel: GAPDH; So, soft hydrogels; St, stiff hydrogels; PS, control polystyrene substrates). Indicated groups were treated with 50 μM PD98059, 10 μM Y27632, or 0.5 μg/ml C3 toxin. Quantification was performed using scanning densitometry. The expression levels of BSP (B and E) and OCN (C and F) were normalized to levels of GAPDH for each condition at both timepoints (B and C: day 1; E and F: day 14), and the ratios were compared with that of wildtype cells on the soft hydrogel at day 1 (arbitrarily assigned a value of 1). Data represent mean ± SD [N ≥ 3; ^a p < 0.05, ^b p < 0.01, and ^c p < 0.001 relative to levels on soft hydrogels; (B) ^d p < 0.05 vs. control cells; (E and F) ^e p < 0.01 vs. control wildtype and V14-RhoA expressing cells].