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• W2072384133 abstract "•Mechanoresponse in multipotent human mammary progenitors is age dependent•The mechanosensing systems are equally active in young and old progenitors•Hippo pathway transducers YAP and TAZ exhibit age-dependent activation efficiencies•YAP localization changes with age in vivo Dysfunctional progenitor and luminal cells with acquired basal cell properties accumulate during human mammary epithelial aging for reasons not understood. Multipotent progenitors from women aged <30 years were exposed to a physiologically relevant range of matrix elastic modulus (stiffness). Increased stiffness causes a differentiation bias towards myoepithelial cells while reducing production of luminal cells and progenitor maintenance. Lineage representation in progenitors from women >55 years is unaffected by physiological stiffness changes. Efficient activation of Hippo pathway transducers YAP and TAZ is required for the modulus-dependent myoepithelial/basal bias in younger progenitors. In older progenitors, YAP and TAZ are activated only when stressed with extraphysiologically stiff matrices, which bias differentiation towards luminal-like phenotypes. In vivo YAP is primarily active in myoepithelia of younger breasts, but localization and activity increases in luminal cells with age. Thus, aging phenotypes of mammary epithelia may arise partly because alterations in Hippo pathway activation impair microenvironment-directed differentiation and lineage specificity. Dysfunctional progenitor and luminal cells with acquired basal cell properties accumulate during human mammary epithelial aging for reasons not understood. Multipotent progenitors from women aged <30 years were exposed to a physiologically relevant range of matrix elastic modulus (stiffness). Increased stiffness causes a differentiation bias towards myoepithelial cells while reducing production of luminal cells and progenitor maintenance. Lineage representation in progenitors from women >55 years is unaffected by physiological stiffness changes. Efficient activation of Hippo pathway transducers YAP and TAZ is required for the modulus-dependent myoepithelial/basal bias in younger progenitors. In older progenitors, YAP and TAZ are activated only when stressed with extraphysiologically stiff matrices, which bias differentiation towards luminal-like phenotypes. In vivo YAP is primarily active in myoepithelia of younger breasts, but localization and activity increases in luminal cells with age. Thus, aging phenotypes of mammary epithelia may arise partly because alterations in Hippo pathway activation impair microenvironment-directed differentiation and lineage specificity. The aging process is often correlated with changes in stem cell activity with consequences ranging from reduced regenerative capacity to increased cancer incidence. Human hematopoietic stem cells accumulate with age (Kuranda et al., 2011Kuranda K. Vargaftig J. de la Rochere P. Dosquet C. Charron D. Bardin F. Tonnelle C. Bonnet D. Goodhardt M. Age-related changes in human hematopoietic stem/progenitor cells.Aging Cell. 2011; 10: 542-546Crossref PubMed Scopus (108) Google Scholar, Pang et al., 2011Pang W.W. Price E.A. Sahoo D. Beerman I. Maloney W.J. Rossi D.J. Schrier S.L. Weissman I.L. Human bone marrow hematopoietic stem cells are increased in frequency and myeloid-biased with age.Proc. Natl. Acad. Sci. USA. 2011; 108: 20012-20017Crossref PubMed Scopus (500) Google Scholar) and exhibit a differentiation bias toward defective myeloid lineages (Cho et al., 2008Cho R.H. Sieburg H.B. Muller-Sieburg C.E. A new mechanism for the aging of hematopoietic stem cells: aging changes the clonal composition of the stem cell compartment but not individual stem cells.Blood. 2008; 111: 5553-5561Crossref PubMed Scopus (244) Google Scholar), making individuals more prone to autoimmune problems and myeloid leukemias (Henry et al., 2011Henry C.J. Marusyk A. DeGregori J. Aging-associated changes in hematopoiesis and leukemogenesis: what’s the connection?.Aging (Albany, N.Y. Online). 2011; 3: 643-656PubMed Google Scholar). In mice, the proportion of mitotic neural stem cells increases with age, whereas numbers of adult-born neurons decrease (Stoll et al., 2011Stoll E.A. Habibi B.A. Mikheev A.M. Lasiene J. Massey S.C. Swanson K.R. Rostomily R.C. Horner P.J. Increased re-entry into cell cycle mitigates age-related neurogenic decline in the murine subventricular zone.Stem Cells. 2011; 29: 2005-2017Crossref PubMed Scopus (22) Google Scholar). Human hippocampus shows patterns of age-related changes similar to mice that may underlie age-related cognitive decline (Knoth et al., 2010Knoth R. Singec I. Ditter M. Pantazis G. Capetian P. Meyer R.P. Horvat V. Volk B. Kempermann G. Murine features of neurogenesis in the human hippocampus across the lifespan from 0 to 100 years.PLoS ONE. 2010; 5: e8809Crossref PubMed Scopus (466) Google Scholar). Transit amplifying cells, not stem cells, accumulate in epidermis with age and delay wound healing (Charruyer et al., 2009Charruyer A. Barland C.O. Yue L. Wessendorf H.B. Lu Y. Lawrence H.J. Mancianti M.L. Ghadially R. Transit-amplifying cell frequency and cell cycle kinetics are altered in aged epidermis.J. Invest. Dermatol. 2009; 129: 2574-2583Crossref PubMed Scopus (39) Google Scholar). Mammary epithelium is maintained by a hierarchy of lineage-biased and multipotent progenitor and stem cells (Nguyen et al., 2014Nguyen L.V. Makarem M. Carles A. Moksa M. Kannan N. Pandoh P. Eirew P. Osako T. Kardel M. Cheung A.M. et al.Clonal analysis via barcoding reveals diverse growth and differentiation of transplanted mouse and human mammary stem cells.Cell Stem Cell. 2014; 14: 253-263Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, Rios et al., 2014Rios A.C. Fu N.Y. Lindeman G.J. Visvader J.E. In situ identification of bipotent stem cells in the mammary gland.Nature. 2014; 506: 322-327Crossref PubMed Scopus (366) Google Scholar, Villadsen et al., 2007Villadsen R. Fridriksdottir A.J. Rønnov-Jessen L. Gudjonsson T. Rank F. LaBarge M.A. Bissell M.J. Petersen O.W. Evidence for a stem cell hierarchy in the adult human breast.J. Cell Biol. 2007; 177: 87-101Crossref PubMed Scopus (291) Google Scholar). In human mammary gland, differentiation-defective cKit-expressing multipotent progenitors (MPPs) accumulate with age, and proportions of daughter myoepithelial (MEP) and luminal epithelial (LEP) cells shift with age. We hypothesized that these age-associated changes make aged breast tissue susceptible to malignant progression (Garbe et al., 2012Garbe J.C. Pepin F. Pelissier F.A. Sputova K. Fridriksdottir A.J. Guo D.E. Villadsen R. Park M. Petersen O.W. Borowsky A.D. et al.Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia.Cancer Res. 2012; 72: 3687-3701Crossref PubMed Scopus (68) Google Scholar). Accumulation of defective stem or progenitor cells may be a common phenotype among aging tissues, and we hypothesize that aged MPPs accumulate because they do not correctly perceive microenvironmental differentiation cues. The molecular composition of microenvironments impose specific cell fate decisions in normal and immortal nonmalignant mammary MPP (LaBarge et al., 2009LaBarge M.A. Nelson C.M. Villadsen R. Fridriksdottir A. Ruth J.R. Stampfer M.R. Petersen O.W. Bissell M.J. Human mammary progenitor cell fate decisions are products of interactions with combinatorial microenvironments.Integr. Biol. (Camb.). 2009; 1: 70-79Crossref PubMed Scopus (143) Google Scholar). Cell culture substrata tuned to elastic moduli that mimicked normal breast tissue also biased the differentiation of an immortal nonmalignant MPP cell line into LEP (Lui et al., 2012Lui C. Lee K. Nelson C.M. Matrix compliance and RhoA direct the differentiation of mammary progenitor cells.Biomech. Model. Mechanobiol. 2012; 11: 1241-1249Crossref PubMed Scopus (32) Google Scholar). Matrix stiffness is mechanistically important in breast cancer progression as well; rigid breast tissue correlates with high breast cancer risk and drives malignant phenotypes in breast cancer cell lines (Yu et al., 2011Yu H. Mouw J.K. Weaver V.M. Forcing form and function: biomechanical regulation of tumor evolution.Trends Cell Biol. 2011; 21: 47-56Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). The physiological range of elastic modulus in breast likely plays an instructive role in the differentiation of normal mammary epithelial progenitors. Membrane and cytoskeleton proteins sense mechanical cues and trigger transduction cascades that relay information throughout the cytoskeleton and to the nucleus. Responses can include changes in morphology and gene expression (Vogel and Sheetz, 2006Vogel V. Sheetz M. Local force and geometry sensing regulate cell functions.Nat. Rev. Mol. Cell Biol. 2006; 7: 265-275Crossref PubMed Scopus (1736) Google Scholar). Sensing matrix elasticity occurs through cell-cell and cell-extracellular matrix (ECM) interactions mediated by adherens, integrins, vinculin, focal adhesion kinase (FAK), and others (Beningo et al., 2001Beningo K.A. Dembo M. Kaverina I. Small J.V. Wang Y.L. Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts.J. Cell Biol. 2001; 153: 881-888Crossref PubMed Scopus (586) Google Scholar, Bershadsky et al., 2003Bershadsky A.D. Balaban N.Q. Geiger B. Adhesion-dependent cell mechanosensitivity.Annu. Rev. Cell Dev. Biol. 2003; 19: 677-695Crossref PubMed Scopus (689) Google Scholar, Tamada et al., 2004Tamada M. Sheetz M.P. Sawada Y. Activation of a signaling cascade by cytoskeleton stretch.Dev. Cell. 2004; 7: 709-718Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). The actinomyosin network includes RhoA, which regulates the actin cytoskeleton in the formation of stress fibers (SFs) and focal adhesions (FAs). Activation of ROCK1/ROCK2 causes increased activity of the motor protein myosin II by phosphorylation of the myosin light chain (MLC) and inactivation of the MLC phosphatase (Ishizaki et al., 1997Ishizaki T. Naito M. Fujisawa K. Maekawa M. Watanabe N. Saito Y. Narumiya S. p160ROCK, a Rho-associated coiled-coil forming protein kinase, works downstream of Rho and induces focal adhesions.FEBS Lett. 1997; 404: 118-124Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar, Kimura et al., 1996Kimura K. Ito M. Amano M. Chihara K. Fukata Y. Nakafuku M. Yamamori B. Feng J. Nakano T. Okawa K. et al.Regulation of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase).Science. 1996; 273: 245-248Crossref PubMed Scopus (2412) Google Scholar). YAP and TAZ are Hippo pathway transcriptional coactivators that are thought to interact with the Rho pathway to transduce mechanical information about the microenvironment to the nucleus (Halder et al., 2012Halder G. Dupont S. Piccolo S. Transduction of mechanical and cytoskeletal cues by YAP and TAZ.Nat. Rev. Mol. Cell Biol. 2012; 13: 591-600Crossref PubMed Scopus (628) Google Scholar). As stiffness increases, YAP/TAZ relocates from cytoplasm into nucleus, where they generate gene expression patterns that underlie cellular functions like proliferation, migration, epithelial-to-mesenchymal transition, and differentiation (Dupont et al., 2011Dupont S. Morsut L. Aragona M. Enzo E. Giulitti S. Cordenonsi M. Zanconato F. Le Digabel J. Forcato M. Bicciato S. et al.Role of YAP/TAZ in mechanotransduction.Nature. 2011; 474: 179-183Crossref PubMed Scopus (3048) Google Scholar, Kanai et al., 2000Kanai F. Marignani P.A. Sarbassova D. Yagi R. Hall R.A. Donowitz M. Hisaminato A. Fujiwara T. Ito Y. Cantley L.C. Yaffe M.B. TAZ: a novel transcriptional co-activator regulated by interactions with 14-3-3 and PDZ domain proteins.EMBO J. 2000; 19: 6778-6791Crossref PubMed Scopus (554) Google Scholar, Zhao et al., 2007Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (1982) Google Scholar). Differentiation of mesenchymal stem cells down neurogenic, myogenic, or osteogenic pathways was directed by exposure to a wide range of tissue-relevant elastic moduli, from 100 to ∼40,000 Pa (Engler et al., 2006Engler A.J. Sen S. Sweeney H.L. Discher D.E. Matrix elasticity directs stem cell lineage specification.Cell. 2006; 126: 677-689Abstract Full Text Full Text PDF PubMed Scopus (9925) Google Scholar). In comparison, mammary MPP differentiation should be responsive to a much narrower range of modulus relevant to normal and malignant breast (100∼4,000 Pa; Paszek et al., 2005Paszek M.J. Zahir N. Johnson K.R. Lakins J.N. Rozenberg G.I. Gefen A. Reinhart-King C.A. Margulies S.S. Dembo M. Boettiger D. et al.Tensional homeostasis and the malignant phenotype.Cancer Cell. 2005; 8: 241-254Abstract Full Text Full Text PDF PubMed Scopus (2800) Google Scholar). The impact of aging on modulus-directed differentiation is unknown. Addressing these issues required a culture-based platform for functional analyses of primary normal human mammary MPP from many individuals. Here, we used such an approach to demonstrate that differentiation patterns of MPP from women aged <30 years cultured on tunable 2D and 3D substrata were exquisitely responsive to a physiologically relevant range of elastic modulus in a YAP/TAZ-dependent manner, whereas MPP from women >55 years were relatively unresponsive to changes in rigidity due to inefficient activation of the Hippo pathway transducers. To test whether microenvironment rigidity directed differentiation in MPP, we enriched receptor tyrosine kinase cKit-expressing (cKit+) human mammary epithelial cells (HMECs) by flow cytometry (fluorescence-activated cell sorting [FACS]) from fourth passage primary prestasis HMEC (strains) derived from five women aged <30 years and five from women >55 years (Figure S1; Table S1). Prestasis HMEC strains are normal and not treated with any immortalizing agents, have finite lifespans, and we previously demonstrated that they retain molecular, biochemical, and functional properties consistent with chronological aging in vivo (Garbe et al., 2012Garbe J.C. Pepin F. Pelissier F.A. Sputova K. Fridriksdottir A.J. Guo D.E. Villadsen R. Park M. Petersen O.W. Borowsky A.D. et al.Accumulation of multipotent progenitors with a basal differentiation bias during aging of human mammary epithelia.Cancer Res. 2012; 72: 3687-3701Crossref PubMed Scopus (68) Google Scholar). The cKit+ MPP were cultured for 48 hr on type 1 collagen-coated polyacrylamide (PA) gels. The Young’s elastic modulus (E[Pa]scals) of the PA gels was tuned from 200 Pa to 2,350 Pa. The lineage of each daughter cell was confirmed by immunofluorescence (IF) of intermediate filament proteins keratin (K)14 and K19, CD227 (sialomucin-1), and CD10 (Calla; Figures 1A, 1B, 1E, and 1F ). Computer image analysis identified the different lineages; LEP are CD227+/CD10−/K14−/K19+, MEP are CD227−/CD10+/K14+/K19−, and K14+K19+ expression is consistent with MPP states (Villadsen et al., 2007Villadsen R. Fridriksdottir A.J. Rønnov-Jessen L. Gudjonsson T. Rank F. LaBarge M.A. Bissell M.J. Petersen O.W. Evidence for a stem cell hierarchy in the adult human breast.J. Cell Biol. 2007; 177: 87-101Crossref PubMed Scopus (291) Google Scholar). cKit+ MPP from women <30 years generated proportionately more LEP on soft substrata, but generation of MEP increased with higher E (Figures 1C, 1C’, 1G, and S2A). cKit+ MPP from women >55 years did not generate different lineage proportions in response to changes in E (Figures 1D, 1D’, 1H, and S2B). Primary first passage cKit+ MPP from three women <30 years, embedded in tunable 3D hydrogels with some type 1 collagen for 7 days (Figure S3A; Ananthanarayanan et al., 2011Ananthanarayanan B. Kim Y. Kumar S. Elucidating the mechanobiology of malignant brain tumors using a brain matrix-mimetic hyaluronic acid hydrogel platform.Biomaterials. 2011; 32: 7913-7923Crossref PubMed Scopus (226) Google Scholar), gave rise to more LEP at 120 Pa versus more MEP at 3,800 Pa (Figures 1K, 1M, 1M’, and S3B). In contrast, cKit+ MPP from three women >55 years did not display modulus-dependent differentiation patterns (Figures 1L, 1N, 1N’, and S3C). The proportion of K14+/K19+ MPP decreased with elastic modulus on 2D PA gels and in 3D gels <30 years HMEC (Figures S4A–S4C), but no change in proportions of MPP was observed in >55 years HMEC (Figures S4B, S4D, and S4F). These results suggested that differentiation was modulus dependent in younger MPP and that this response was lost with age. To test if changes in lineage proportions were due to lineage-biased proliferation, incorporation of 5-ethynyl-2′-deoxyuridine (EdU) into DNA was measured as a proxy for proliferation. All lineages derived after 48 hr from cKit+ MPP on PA gels exhibited similar proportions of EdU incorporation (EdU+), irrespective of substrate rigidity or age (Figure 1I). In contrast, EdU incorporation in unsorted HMEC strains, which are primarily composed of more mature LEP and MEP, revealed age- and lineage-specific replicative behaviors. Proportions of EdU+ MEP from <30 years HMEC significantly increased with greater E whereas EdU+ LEP trended downward (Figure 1J). Unsorted HMEC from women >55 years exhibited neither lineage- nor modulus-dependent proliferation (Figure 1J), underscoring the lack of mechanoresponse with age. Thus, changes in lineage proportions exhibited by <30 years MPP after 48 hr were likely due to modulus-dependent differentiation, and only after the lineages matured in <30 years HMEC strains, did they show evidence of modulus-dependent proliferation. To determine whether mechanosensing was age dependent, F-actin SF formation and FA assembly activities were measured in cKit+ MPP from three <30 years and three >55 years strains. Irrespective of age, SF formation increased with greater E (Figure 2A). Homogeneity measurements were used to quantify formation of the F-actin cables (Haralick et al., 1973Haralick R.M. Shanmugam K. Dinstein I. Textural features for image classification.IEEE Trans. Syst. Man Cyb. 1973; SMC-3: 610-621Crossref Scopus (16887) Google Scholar, Pantic et al., 2012Pantic I. Pantic S. Basta-Jovanovic G. Gray level co-occurrence matrix texture analysis of germinal center light zone lymphocyte nuclei: physiology viewpoint with focus on apoptosis.Microsc. Microanal. 2012; 18: 470-475Crossref PubMed Scopus (43) Google Scholar); SF homogeneity was inversely proportional to E in both age groups, which showed similar slopes (Figures 2B and 2C). Progenitors from both age groups were stained for pFAK and vinculin, which colocalize at FA. More FA assemblies were observed with increased E at the interfaces of MPP and gels, and FA formation was not impaired with age (Figures 2D and 2E). FA homogeneity was inversely proportional to E with comparable slopes in both age groups (Figures 2F and 2G). Thus, similar SF and FA phenotypes were observed both in younger and older cKit+ MPP. Extracellular signal-regulated kinase (ERK) is phosphorylated in response to increased elastic modulus in some adherent cell lines (Provenzano et al., 2009Provenzano P.P. Inman D.R. Eliceiri K.W. Keely P.J. Matrix density-induced mechanoregulation of breast cell phenotype, signaling and gene expression through a FAK-ERK linkage.Oncogene. 2009; 28: 4326-4343Crossref PubMed Scopus (452) Google Scholar), and because it is a key effector of serum responses, changes in its modulation could cause pleiotropic cellular responses. Both young and old cKit+ MPP exhibited a low level of ERK phosphorylation on 200 Pa PA gels but increased up to 15-fold on 2,350 Pa PA gels (Figures 2H and 2I). Differences in pERK were not significant between age groups, and mitogen-activated protein kinase (MAPK) inhibitor PD98059 prevented ERK phosphorylation. By these measures, modulus-dependent activity in serum response was independent of age. If mechanosensing was unaffected by aging, then perturbations of actinomyosin regulators that are known to alter FA or SF formation should elicit parallel phenotypes in young and old MPP. Independent of age, we observed that inhibitors of ROCK1/ROCK2 (Y27632) and MLC kinase (ML-7) tended to disrupt SF on 2,350 Pa substrata and showed little effect at 200 Pa, whereas the MLC phosphatase inhibitor calyculin A (calA) caused SF formation at 200 Pa (Figures 3A and 3B ). Patterns of changes in F-actin homogeneity were parallel in both age groups (Figures 3C and 3D). Y27632 and ML-7 disrupted the FA assemblies on 2,350 Pa substrata, whereas calA promoted FA assembly on 200 Pa substrata (Figures 3E and 3F) and measurements of FA homogeneity showed parallel changes in both age groups (Figures 3G and 3H). Mechanosensing of substrata in the physiologically relevant range was age independent, evaluated by these measures, and thus was unlikely to account for age-dependent differences in mechanically directed differentiation. Analysis of the effects of actinomyosin network modulation on differentiation in three young and three old cKit+ MPP revealed surprising age-dependent responses. FA and SF in cells treated with Y27632 and ML-7 on 2,350 Pa gels were similar to untreated cells on 200 Pa gels, whereas calA increased the SF and FA as if cells were on stiffer substrata. Thus, SF and FA phenotypes were pharmacologically manipulated irrespective of the actual substrate E. Younger cKit+ MPP treated with Y27632 or ML-7 significantly increased proportions of K19+ LEP compared to DMSO-treated cells on 2,350 Pa gels. In contrast, older MPP were unaffected by Y27632 or ML-7 treatment (Figure S5). Interestingly, calA increased MEP generation from young MPP but caused older MPP to give rise to significantly more LEP. No changes in proportion were observed on 200 Pa gels, suggesting that addition of calA was insufficient to trigger cell differentiation on such a soft substrata. Older progenitors did not respond to chemically decreased perception of stiffness, but they responded to artificially higher stiffness oppositely to that of young progenitors, suggesting that age-dependent transcriptional programs were operative. We next determined whether the mechanotransducive transcription factors YAP and TAZ were involved in modulus-dependent differentiation. IF staining of YAP and TAZ in cKit+ MPP from three HMEC strains <30 years exhibited increased YAP/TAZ nuclear translocation as PA gel E increased from 200 to 2,350 Pa (Figures 4A and 4B ). However, significant nuclear translocation of YAP/TAZ was not observed in older progenitors in the same range (Figures 4C and 4D). The ability to activate YAP/TAZ by changes in substrate E was age dependent. To determine the extent to which YAP/TAZ was unresponsive to changes in E in older progenitors, we evaluated their activation in response to extraphysiological stiffness. cKit+ MPP from three young and three old strains were cultured on type 1 collagen-coated glass (>3 GigaPa) or 200 Pa gels. YAP/TAZ translocated to the nucleus in young cKit+ MPP on glass (Figures 4A and 4B), gave rise to fewer LEP (Figure 4E), and those LEP incorporated less EdU on glass compared to 200 Pa gels (Figure 4F). In older cKit+ MPP on glass, YAP/TAZ was nuclear (Figures 4C and 4D), but cells gave rise to more LEP (Figure 4G), which incorporated more EdU on glass than on 200 Pa gels (Figure 4H). Both calA treatment and culture on extraphysiologically rigid substrata elicited more LEP differentiation in older MPP, which was the opposite of younger MPP. Thus, the Hippo pathway mechanoresponse in older progenitors was shifted to an extraphysiological “trigger point”. To determine if YAP and TAZ were required for modulus-dependent differentiation, both in the physiological and extraphysiological ranges, cKit+ MPP were transfected with small interfering RNAs (siRNAs), siYAP or siTAZ, which achieved >70% knockdown of the respective mRNAs (Figure 4K). K14 and K19 proteins were measured by IF in each cell after 48 hr on PA gels. Younger MPP harboring either siYAP or siTAZ were unable to give rise to more MEP on 2,350 Pa PA gels and glass substrata compared to controls (Figure 4I). In older MPP, siYAP and siTAZ had no effect at 200 Pa or 2,350 Pa, but they prevented the generation of more LEP on glass (Figure 4J). Thus, YAP and TAZ were required for modulus-dependent differentiation in progenitors irrespective of age. That YAP and TAZ activity correlated with a bias toward MEP in younger women prompted us to evaluate normal breast tissue sections from reduction mammoplasty. K14, K19, and YAP were evaluated by IF in sections from four women aged 34 years, 40 years, 50 years, and 54 years (Figure 5A). Multiple fields from each section were analyzed to account for heterogeneity, and marker-based watershed cell segmentation identified levels of YAP in nuclear and cytoplasmic domains of MEP, LEP, and K14+/K19+ putative MPP. YAP staining was localized mainly to the nuclei of MEP and MPP in the 34 years and 40 years glands (Figure 5B). In the 34 years gland, we also observed a number of occurrences of K14+/K19− cells that we assumed were LEP based on their luminal location. Upon measuring the YAP signal intensity, it was determined that the K14+ cells always correlated with higher YAP expression (Figure 5C). In contrast to younger epithelia, the 50 years and 54 years glands exhibited no inequality in YAP localization between the different lineages (Figure 5B), and qualitatively, the even-appearing distribution of YAP in the nuclei and cytoplasm of the LEP and MEP in older women was reminiscent of YAP staining in >55 years HMEC (Figure 5C). YAP distribution in vivo was age dependent consistent with our findings in primary cultures. Overall, the data suggest that YAP activity is associated with MEP/basal phenotypes, even in the case of LEP in older women that acquire some traits of MEP. That impression was strengthened by our analysis of breast cancer data from The Cancer Genome Atlas data (Cancer Genome Atlas, 2012Cancer Genome Atlas N. Cancer Genome Atlas NetworkComprehensive molecular portraits of human breast tumours.Nature. 2012; 490: 61-70Crossref PubMed Scopus (7887) Google Scholar), which showed that YAP/TAZ mRNA expression negatively correlated with levels of LEP-related proteins and mRNAs and positively correlated with markers of MEP and with YAP/TAZ target genes (Figures S6A and S6B). To better understand why the trigger point for YAP/TAZ was increased in older MPP, we determined whether Hippo pathway components showed age-dependent expression patterns. Mst1/Mst2, Lats2, and angiomotins (AMOT) phosphorylate and sequester YAP/TAZ in the cytoplasm, which would prevent YAP/TAZ from associating with DNA-binding cofactors TEAD1–TEAD4 and transcribing target genes, like connective tissue growth factor (CTGF) (Zhao et al., 2007Zhao B. Wei X. Li W. Udan R.S. Yang Q. Kim J. Xie J. Ikenoue T. Yu J. Li L. et al.Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control.Genes Dev. 2007; 21: 2747-2761Crossref PubMed Scopus (1982) Google Scholar, Zhao et al., 2011Zhao B. Li L. Lu Q. Wang L.H. Liu C.Y. Lei Q. Guan K.L. Angiomotin is a novel Hippo pathway component that inhibits YAP oncoprotein.Genes Dev. 2011; 25: 51-63Crossref PubMed Scopus (480) Google Scholar). Analysis with quantitative real-time PCR (qRT-PCR) revealed that, in almost all cases, MST1/MST2, LATS2, AMOT, AMOTL1, TEAD1–TEAD4, and CTGF were significantly correlated between women <30 years and women >55 years on 200 Pa (Figure 6A; p = 0.0110; R = 0.7283) and 2,350 Pa (Figure 6B; p = 0.0083; R = 0.7464) gels. On glass, the correlation was less pronounced (Figure 6C; p = 0.0543; R = 0.5934). Because differences in Hippo gene expression were not strikingly age dependent in physiological conditions, we examined protein levels of MST1 and MST2. MST1 levels from three women <30 years and three women >55 years were not significantly different (Figures 6D–6G). MST2 protein levels were 3-fold greater in MPP from women >55 years compared to <30 years on 2,350 Pa gels (Figure 6E). Thus, it is tempting to speculate that age-dependent stoichiometry of Hippo pathway regulators can lead to insensitivity to mechanical cues and activation of YAP/TAZ. Cellular responses to mechanical stimuli are often examined with mesenchymal stem cells or immortal malignant and nonmalignant cell lines. Whereas immortal cell lines tend to proliferate more on stiffer substrata, we showed that normal <30 years HMEC exhibited lineage-dependent responses and that <55 years HMEC were nonresponsive to a physiological range of E (Figure 1). To better understand the differences between the normal and immortal nonmalignant states as a function of age, we examined immortal nonmalignant cell lines derived from two young and two old primary HMEC strains by targeted inactivation of senescence barriers combined with unknown genomic errors (Stampfer et al., 2013Stampfer M.M. LaBarge M.A. Garbe J.C. An Integrated Human Mammary Epithelial Cell Culture System for Studying Carcinogenesis and Aging.in: Schatten H. Cell and Molecular Biology of Breast Cancer. Springer, New York2013: 323-361Crossref Scopus (23) Google Scholar). The cell lines, 240LMY (19 years), 184Fp16s (21 years), 122LMY (66 years), and 805Pp16s (91 years; Figure S7) were cultured atop PA gels tuned from 200 to 2,350 Pa. cKit+ MPP from 240LMY and 184Fp16s cell lines gave rise to more K14+ MEP than LEP on glass compared to 200 Pa PA gels (Figure 7A). cKit+ MPP from 122LMY and 805Pp16s gave rise to more K19+ LEP than MEP on glass compared to 200 Pa PA gels (Figure 7B). Independent of age, all lineages of the immortal cell lines incorp" @default.
• W2072384133 created "2016-06-24" @default.
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• W2072384133 date "2014-06-01" @default.
• W2072384133 modified "2023-10-16" @default.
• W2072384133 title "Age-Related Dysfunction in Mechanotransduction Impairs Differentiation of Human Mammary Epithelial Progenitors" @default.
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