Commitment of stem cells to different lineages is regulated by many cues in the local tissue microenvironment. Here we demonstrate that cell shape regulates commitment of human mesenchymal stem cells (hMSCs) to adipocyte or osteoblast fate. hMSCs allowed to adhere, flatten, and spread underwent osteogenesis, while unspread, round cells became adipocytes. Cell shape regulated the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed hMSCs to become adipocytes, while constitutively active RhoA caused osteogenesis. However, the RhoA-mediated adipogenesis or osteogenesis was conditional on a round or spread shape, respectively, while constitutive activation of the RhoA effector, ROCK, induced osteogenesis independent of cell shape. This RhoA-ROCK commitment signal required actin-myosin-generated tension. These studies demonstrate that mechanical cues experienced in developmental and adult contexts, embodied by cell shape, cytoskeletal tension, and RhoA signaling, are integral to the commitment of stem cell fate.
The tumour microenvironment can be a potent carcinogen, not only by facilitating cancer progression and activating dormant cancer cells, but also by stimulating tumour formation 1 . We have previously investigated stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours 2 , and found that MMP-3 can cause epithelial-mesenchymal transition (EMT) and malignant transformation in cultured cells [3][4][5] , and genomically unstable mammary carcinomas in transgenic mice 3 . Here we explain the molecular pathways by which MMP-3 exerts these effects: exposure of mouse mammary epithelial cells to MMP-3 induces the expression of an alternatively spliced form of Rac1, which causes an increase in cellular reactive oxygen species (ROS). The ROS stimulate the expression of the transcription factor Snail and EMT, and cause oxidative damage to DNA and genomic instability. These findings identify a previously undescribed pathway in which a component of the breast tumour microenvironment alters cellular structure in culture and tissue structure in vivo, leading to malignant transformation.Cancer is characterized by a progressive series of alterations that disrupt cell and tissue homeostasis. Whereas many of these alterations can be induced by specific mutations, faulty signals from the microenvironment also can act as inducers of tumour development and progression 1 . MMPs are prominent contributors to such micro-environmental signals, because these proteolytic enzymes degrade structural components of the extracellular matrix (ECM), permitting tumour invasion and metastasis. Additionally, MMPs can release cell-bound inactive precursor forms of growth factors, degrade cell-cell and cell-ECM adhesion molecules, activate precursor zymogen forms of other MMPs, and inactivate inhibitors of MMPs and other proteases 6 . Our observations that MMP-3 can induce transformation in mammary epithelial cells in culture 4,5,7 Supplementary Fig. 1a,b), through a process that is initially reversible (refs 4, 5; Supplementary Fig. 1c). The MMP-3-induced alteration of the F-actin cytoskeleton (Fig. 1a) indicated the possible involvement of members of the Rho GTPase family, and although the activities of RhoA and Cdc42 were unchanged (not shown), we were intrigued by an additional band in the Rac activity assay of MMP-3-treated cells (Fig. 1b). A highly activated splice isoform of Rac1, designated Rac1b, containing 57 additional nucleotides that result in an in-frame insertion of 19 additional amino acid residues, was discovered recently in breast and colorectal tumours 8,9 and has transforming characteristics when exogenously expressed in cultured cells 10 . We identified the additional Rac band induced by MMP-3 as Rac1b by polymerase chain reaction with reverse transcription (RT-PCR) (Fig. 1c) and through the use of an antibody raised against the mouse Rac1b insertion sequence (Fig. 1d); we also found that induction of Rac1b by treatment with MMP-3 was initially reversible (Fig. 1e). We determi...
The microenvironment influences gene expression so that the behavior of a cell is largely determined by its interactions with the extracellular matrix, neighboring cells, and soluble local and systemic cues. We describe the essential roles of context and organ structure in directing mammary gland development and differentiated function and in determining the response to oncogenic insults, including mutations. We expand on the concept of "dynamic reciprocity" to present an integrated view of development, cancer, and aging and posit that genes are like the keys on a piano: Although they are essential, it is the context that makes the music.
Spatial patterns of cellular growth generate mechanical stresses that help to push, fold, expand, and deform tissues into their specific forms. Genetic factors are thought to specify patterns of growth and other behaviors to drive morphogenesis. Here, we show that tissue form itself can feed back to regulate patterns of proliferation. Using microfabrication to control the organization of sheets of cells, we demonstrated the emergence of stable patterns of proliferative foci. Regions of concentrated growth corresponded to regions of high tractional stress generated within the sheet, as predicted by a finite-element model of multicellular mechanics and measured directly by using a micromechanical force sensor array. Inhibiting actomyosin-based tension or cadherin-mediated connections between cells disrupted the spatial pattern of proliferation. These findings demonstrate the existence of patterns of mechanical forces that originate from the contraction of cells, emerge from their multicellular organization, and result in patterns of growth. Thus, tissue form is not only a consequence but also an active regulator of tissue growth. Spatial patterning of the behaviors of individual cells generates global changes in tissue architecture that drive morphogenesis (1, 2). Several morphogenic mechanisms likely collaborate to direct tissue form, including local changes in cell adhesion, cell shape, and cell proliferation. Qualitative and quantitative differences in cellular adhesiveness can lead to the segregation and layering of tissues (3); ordered changes in cell shape appear to direct gastrulation (4), epithelial folding (5), and tubulogenesis (6); and differentials in cell growth can locally alter tissue form (7,8). Although the molecular basis for these behaviors has been under intense study, the mechanical nature of morphogenesis also has been recognized since the late 19th century (9): Specific patterns of cellular growth (in which some cells proliferate but other cells do not) create mechanical stresses that help drive the buckling, budding, pinching, and branching processes of morphogenesis. Complex forms, such as the regular fractal structure of the branching organs, can thus arise from simple embryonic sheets (reviewed in refs. 10 and 11).What causes such localized patterns is one of the central puzzles of biology and has fascinated scientists from numerous disciplines for at least two millennia (12). Perhaps most well described are concentration gradients of diffusible factors, known as morphogens, which can drive spatial patterns of cellular behaviors (13-15). In addition to soluble factors, adhesion to extracellular matrix and mechanical forces also are known to modulate cell functions, including proliferation (10,16,17).Although spatial patterning of these cues can certainly explain spatial patterning of cellular behaviors, it remains unclear what initiates or maintains patterns. One theory suggests that these gradients (e.g., of morphogens) are entirely driven by prespecified genetic programs. A more tracta...
Actomyosin contractility affects cellular organization within tissues in part through the generation of mechanical forces at sites of cell-matrix and cell-cell contact. While increased mechanical loading at cell-matrix adhesions results in focal adhesion growth, whether forces drive changes in the size of cell-cell adhesions remains an open question. To investigate the responsiveness of adherens junctions (AJ) to force, we adapted a system of microfabricated force sensors to quantitatively report cell-cell tugging force and AJ size. We observed that AJ size was modulated by endothelial cell-cell tugging forces: AJs and tugging force grew or decayed with myosin activation or inhibition, respectively. Myosin-dependent regulation of AJs operated in concert with a Rac1, and this coordinated regulation was illustrated by showing that the effects of vascular permeability agents (S1P, thrombin) on junctional stability were reversed by changing the extent to which these agents coupled to the Rac and myosin-dependent pathways. Furthermore, direct application of mechanical tugging force, rather than myosin activity per se, was sufficient to trigger AJ growth. These findings demonstrate that the dynamic coordination of mechanical forces and cell-cell adhesive interactions likely is critical to the maintenance of multicellular integrity and highlight the need for new approaches to study tugging forces.adherens junction | mechanotransduction | myosin | PDMS | traction force
The treelike structures of many organs, including the mammary gland, are generated by branching morphogenesis, a reiterative process of branch initiation and invasion from a preexisting epithelium. Using a micropatterning approach to control the initial three-dimensional structure of mouse mammary epithelial tubules in culture, combined with an algorithm to quantify the extent of branching, we found that the geometry of tubules dictates the position of branches. We predicted numerically and confirm experimentally that branches initiate at sites with a local minimum in the concentration of autocrine inhibitory morphogens, such as transforming growth factor-β. These results reveal that tissue geometry can control organ morphogenesis by defining the local cellular microenvironment, a finding that has relevance to control of invasion and metastasis.A burst of dichotomous and lateral branching at puberty transforms the mammary epithelial tubule rudiment present at birth into a fully elaborated ductal tree in the female adult. The overall process of branching morphogenesis is regulated globally by a number of cues, including growth factors, extracellular matrix (ECM) molecules, proteases, and morphogens (1-4). These global cues must be integrated locally within the context of the tissue to determine where branches are initiated; thus, a subgroup of epithelial cells is instructed to form a branch or to bifurcate, whereas neighboring cells are not (5). Current techniques to study this process, which is common to many organs including the lung, kidney, and salivary gland, do not allow for a precise quantitative understanding of how spatial positioning is determined. Given that the mammary ductal network branches out from preexisting epithelial tubules, we hypothesized that the position of cells within a tubule might provide contextual information to instruct branch site initiation.To define the role of positional context, we developed a three-dimensional (3D) micropatterned assay for mammary epithelial branching morphogenesis that allowed us to mimic the mammary rudiment by controlling the initial geometry of tubules and to quantify the positions at which they branched. We engineered epithelial tubules of defined geometry by embedding functionally normal mouse mammary epithelial (EpH4) cells in cavities of collagen gel generated by molding unpolymerized collagen I around a patterned elastomeric stamp (Fig. 1A) (6). Embedded epithelial cells formed hollow tubules (7) conforming to the size and shape of the collagen cavities (Fig. 1, B NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript its magnitude and position statistically, we stained nuclei and stacked multiple fluorescent images in registration such that the stacked image revealed the average spatial distribution of cells within, and branching from, the tubules (Fig. 1D). Stacked images were depicted as frequency maps ( Fig. 1E) (8).The tubules remained quiescent ( Fig. 1E) until induced to undergo branching morphogenesis by addition o...
Background Mendelian analysis of disorders of immune regulation can provide insight into molecular pathways associated with host defense and immune tolerance. Methods We identified three families with a dominantly inherited complex of cold-induced urticaria, antibody deficiency, and susceptibility to infection and autoimmunity. Immunophenotyping methods included flow cytometry, analysis of serum immunoglobulins and autoantibodies, lymphocyte stimulation, and enzymatic assays. Genetic studies included linkage analysis, targeted Sanger sequencing, and next-generation whole-genome sequencing. Results Cold urticaria occurred in all affected subjects. Other, variable manifestations included atopy, granulomatous rash, autoimmune thyroiditis, the presence of antinuclear antibodies, sinopulmonary infections, and common variable immunodeficiency. Levels of serum IgM and IgA and circulating natural killer cells and class-switched memory B cells were reduced. Linkage analysis showed a 7-Mb candidate interval on chromosome 16q in one family, overlapping by 3.5 Mb a disease-associated haplotype in a smaller family. This interval includes PLCG2, encoding phospholipase Cγ2 (PLCγ2), a signaling molecule expressed in B cells, natural killer cells, and mast cells. Sequencing of complementary DNA revealed heterozygous transcripts lacking exon 19 in two families and lacking exons 20 through 22 in a third family. Genomic sequencing identified three distinct in-frame deletions that cosegregated with disease. These deletions, located within a region encoding an autoinhibitory domain, result in protein products with constitutive phospholipase activity. PLCG2-expressing cells had diminished cellular signaling at 37°C but enhanced signaling at subphysiologic temperatures. Conclusions Genomic deletions in PLCG2 cause gain of PLCγ2 function, leading to signaling abnormalities in multiple leukocyte subsets and a phenotype encompassing both excessive and deficient immune function. (Funded by the National Institutes of Health Intramural Research Programs and others.)
In order to understand why cancer develops as well as predict the outcome of pharmacological treatments, we need to model the structure and function of organs in culture so that our experimental manipulations occur under physiological contexts. This review traces the history of the development of a prototypic example, the three-dimensional (3D) model of the mammary gland acinus. We briefly describe the considerable information available on both normal mammary gland function and breast cancer generated by the current model and present future challenges that will require an increase in its complexity. We propose the need for engineered tissues that faithfully recapitulate their native structures to allow a greater understanding of tissue function, dysfunction, and potential therapeutic intervention.
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