Nuclear Lamin-A Scales with Tissue Stiffness and Enhances Matrix-Directed Differentiation

Introduction Tissues can be soft like brain, bone marrow, and fat, which bear little mechanical stress, or stiff like muscle, cartilage, and bone, which sustain high levels of stress. Systematic relationships between tissue stiffness, protein abundance, and differential gene expression are unclear. Recent studies of stem cells cultured on matrices of different elasticity, E, have suggested that differentiation is mechanosensitive, but the molecular mechanisms involved in particular tissues remain elusive. Tissue micromechanics correlate with abundance of collagens and nuclear lamins, which influence cell differentiation. (Left) Collagen and lamin-A levels scale with E, consistent with matching tissue stress to nuclear mechanics. (Right) Matrix stiffness in tissue culture increases cell tension and stabilizes lamin-A, regulating its own transcription and that of stress fiber genes, enhancing differentiation. RA, retinoic acid, i.e., vitamin A; RARG, YAP1, and SRF, transcription factors. Methods We developed quantitative mass spectrometry algorithms to measure protein abundance, stoichiometry, conformation, and interactions within tissues and cells in relation to stiffness of tissues and extracellular matrix. Manipulations of lamin-A levels with small interfering RNA, overexpression, and retinoic acid or antagonist were applied to stem cells cultured on different matrices to assess lamin-A’s role in mechanosensitive differentiation. To characterize molecular mechanisms, promoter analyses, transcriptional profiling, and localization of transcription factors were complemented by measurements of nuclear mechanics and by modeling of the core gene circuit. Results Proteomic profiling of multiple adult solid tissues showed that widely varied levels of collagens in extracellular matrix and of lamin-A in nuclei followed power-law scaling versus E. Scaling for mechanoresponsive lamin-A conformed to predictions from polymer physics, whereas lamin-B’s varied weakly. Tumor xenograft studies further demonstrated that matrix determined tissue E, whereas lamin-A levels responded to changes in E. In tissue culture cells, both lamin-A conformation and expression were mechanosensitive, with phosphorylation and turnover of lamin-A correlating inversely with matrix E. Lamin-A knockdown enhanced mesenchymal stem cell differentiation on soft matrix that favored a low-stress, fat phenotype. Lamin-A overexpression or transcriptional induction with a retinoic acid (RA) antagonist enhanced differentiation on stiff matrix toward a high-stress, bone phenotype. Downstream of matrix stiffness, the RA pathway regulated lamin-A transcription, but feedback by lamin-A regulated RA receptor (RARG) translocation into nuclei. High lamin-A levels physically impeded nuclear remodeling under stress but also coregulated other key factors. These factors included both serum response factor (SRF), which promoted expression of stress fiber–associated proteins involved in differentiation, and a Hippo pathway factor (YAP1) involved in growth. Discussion The characteristic stress in normal tissue favors collagen accumulation and a characteristic stiffness that cells transduce through nuclear lamin-A to enhance tissue-specific differentiation. Tension-inhibited turnover of rope-like filaments of lamin-A provides sufficient mechanochemical control of a core gene circuit to explain the steady-state scaling of lamin-A with E. High lamin-A physically stabilizes the nucleus against stress and thereby stabilizes the nuclear lamina and chromatin, with implications for epigenetic stabilization and limiting of DNA breaks. Moreover, lamin-A levels directly or indirectly regulate many proteins involved in tissue-specific gene expression, and, because lamin-A levels can vary by a factor of 10 or more downstream of tissue mechanics, an important fraction of tissue-specific gene expression depends on tissue mechanics, which changes in development, injury, and many diseases. Lamins and Tissue Stiffness Microenvironment can influence cell fate and behavior; for example, extracellular matrix (ECM) stiffness increases cell proliferation, and ECM rigidity induces disorders in tissue morphogenesis by increasing cell tension. Swift et al. (1240104; see the Perspective by Bainer and Weaver) used proteomics to identify molecules that are mechanical sensors for tissue elasticity in the nucleus and discovered that expression of lamin-A levels apparently functions as a “mechanostat. ” Tissues that need to remain stiff under stress rely on lamin-A to keep the cell nucleus whole. [Also see Perspective by Bainer and Weaver] Tissues can be soft like fat, which bears little stress, or stiff like bone, which sustains high stress, but whether there is a systematic relationship between tissue mechanics and differentiation is unknown. Here, proteomics analyses revealed that levels of the nucleoskeletal protein lamin-A scaled with tissue elasticity, E, as did levels of collagens in the extracellular matrix that determine E. Stem cell differentiation into fat on soft matrix was enhanced by low lamin-A levels, whereas differentiation into bone on stiff matrix was enhanced by high lamin-A levels. Matrix stiffness directly influenced lamin-A protein levels, and, although lamin-A transcription was regulated by the vitamin A/retinoic acid (RA) pathway with broad roles in development, nuclear entry of RA receptors was modulated by lamin-A protein. Tissue stiffness and stress thus increase lamin-A levels, which stabilize the nucleus while also contributing to lineage determination.