FIGURE 5:

Influence of soluble factors on RA pathway implicated in osteogenesis: convergence of mechanochemical effects. (A) MSCs cultured on plastic with drugs that perturb the RA pathway (control is ethanol/dimethyl sulfoxide vehicle only; drug treatments at 1 µM, except adapalene and CD437 at 0.1 µM; see Supplemental Figure S4A for summary of drug properties). LMNA was quantified by immunostaining after 4 d. (B) Although the magnitude of response varied across MSCs from different donors, RA pathway antagonists consistently increased LMNA level with respect to the agonist on a range of stiff substrates (rigid plastic, stiff polyacrylamide gels, collagen thin films; see Supplemental Figure S4B for full table); iPS-derived MSCs and progeria patient–derived iPS-MSCs gave similar response (bottom of the table). (C) Antagonist/agonist effects on LMNA levels from immunofluorescence of iPS-MSCs and progeria patient–derived iPS-MSCs relative to control. The immunoblots (bottom) showed that the progerin from the disease-causing allele was affected as much as the normal lamin-A spliceforms. (D) LMNA was quantified by immunofluorescence in MSCs treated for 3 d with RARG-specific RA pathway antagonist CD2665 or agonist CD1530 while being cultured on pristine or cross-linked collagen-I films. Consistent with earlier reports (Swift et al., 2013b), a significant differential response between agonist and antagonist is apparent only on a “stiff” (cross-linked) substrate. (E) (i) The spread area of nuclei in iPS-progeria–derived MSCs depends on matrix stiffness and is reversible. Early passage, P1, MSCs spread less as P2 on soft gels than on stiff gels or plastic. (F) Nuclear area and lamin-A in iPS-progeria–derived MSCs on soft gels of 2 kPa shows that 95% of cells are within ±2σ of untreated cells even after treatment with RA agonist/antagonist. These cells are “weak responders” to retinoids, and the same range of small nuclear areas identifies weak responders to retinoids on stiff gels of 50 kPa. However, about one-third of cells are mechanosensitive to stiffness, with elevated lamin-A levels that also change in response to retinoids. (ii) Nuclear RARG in bone marrow–derived MSCs on pristine and cross-linked films increases (for responding cells) with matrix stiffness, but retinoids have no effect on RARG, even though lamin-A tends to decrease with agonist and increase with antagonist.