Mechanical Stabilization of the Glandular Acinus by Linker of Nucleoskeleton and Cytoskeleton Complex.

The nucleoskeleton and cytoskeleton are important protein networks that govern cellular behavior and are connected together by the linker of nucleoskeleton and cytoskeleton (LINC) complex. Mutations in LINC complex components may be relevant to cancer, but how cell-level changes might translate into tissue-level malignancy is unclear. We used glandular epithelial cells in a three-dimensional culture model to investigate the effect of perturbations of the LINC complex on higher order cellular architecture. We show that inducible LINC complex disruption in human mammary epithelial MCF-10A cells and canine kidney epithelial MDCK II cells mechanically destabilizes the acinus. Lumenal collapse occurs because the acinus is unstable to increased mechanical tension that is caused by upregulation of Rho-kinase-dependent non-muscle myosin II motor activity. These findings provide a potential mechanistic explanation for how disruption of LINC complex may contribute to a loss of tissue structure in glandular epithelia.

N-cadherin adhesive interactions modulate matrix mechanosensing and fate commitment of mesenchymal stem cells.

During mesenchymal development, the microenvironment gradually transitions from one that is rich in cell-cell interactions to one that is dominated by cell-ECM (extracellular matrix) interactions. Because these cues cannot readily be decoupled in vitro or in vivo, how they converge to regulate mesenchymal stem cell (MSC) mechanosensing is not fully understood. Here, we show that a hyaluronic acid hydrogel system enables, across a physiological range of ECM stiffness, the independent co-presentation of the HAVDI adhesive motif from the EC1 domain of N-cadherin and the RGD adhesive motif from fibronectin. Decoupled presentation of these cues revealed that HAVDI ligation (at constant RGD ligation) reduced the contractile state and thereby nuclear YAP/TAZ localization in MSCs, resulting in altered interpretation of ECM stiffness and subsequent changes in downstream cell proliferation and differentiation. Our findings reveal that, in an evolving developmental context, HAVDI/N-cadherin interactions can alter stem cell perception of the stiffening extracellular microenvironment.

The mechanical regulation of integrin-cadherin crosstalk organizes cells, signaling and forces.

Cadherins and integrins are intrinsically linked through the actin cytoskeleton and share common signaling molecules. Although mechanosensing by the integrin-actin axis has long been appreciated, a growing body of literature now demonstrates that cadherins also transduce and respond to mechanical forces. Mounting evidence shows that mechanically driven crosstalk between integrins and cadherins regulates the spatial distribution of these receptors, their signaling intermediates, the actin cytoskeleton and intracellular forces. This interplay between integrins and cadherins can control fibronectin matrix assembly and signaling, and a fine balance between traction forces at focal adhesions and intercellular tension at adherens junctions is crucial for directional collective cell migration. In this Commentary, we discuss two central ideas: (1) how the dynamic interplay between integrins and cadherins regulates the spatial organization of intracellular signals and the extracellular matrix, and (2) the emerging consensus that intracellular force is a central mechanism that dictates cell behavior, guides tissue development and ultimately drives physiology.

Apolipoprotein E3 Inhibits Rho to Regulate the Mechanosensitive Expression of Cox2.

Apolipoprotein E3 (apoE3) is thought to protect against atherosclerosis by enhancing reverse cholesterol transport. However, apoE3 also has cholesterol-independent effects that contribute to its anti-atherogenic properties. These include altering extracellular matrix protein synthesis and inhibiting vascular smooth muscle cell proliferation. Both of these cholesterol-independent effects result from an apoE3-mediated induction of cyclooxygenase-2 (Cox2). Nevertheless, how apoE3 regulates Cox2 remains unknown. Here, we show that apoE3 inhibits the activation of Rho, which reduces the formation of actin stress fibers and focal adhesions and results in cellular softening. Inhibition of Rho-Rho kinase signaling or direct cellular softening recapitulates the effect of apoE3 on Cox2 expression while a constitutively active Rho mutant overrides the apoE3 effect on both intracellular stiffness and Cox2. Thus, our results describe a previously unidentified mechanism by which an atheroprotective apolipoprotein uses Rho to control cellular mechanics and Cox2.

N-Cadherin Induction by ECM Stiffness and FAK Overrides the Spreading Requirement for Proliferation of Vascular Smooth Muscle Cells.

In contrast to the accepted pro-proliferative effect of cell-matrix adhesion, the proliferative effect of cadherin-mediated cell-cell adhesion remains unresolved. Here, we studied the effect of N-cadherin on cell proliferation in the vasculature. We show that N-cadherin is induced in smooth muscle cells (SMCs) in response to vascular injury, an in vivo model of tissue stiffening and proliferation. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated activation of a focal adhesion kinase (FAK)-p130Cas-Rac signaling pathway induces N-cadherin. Additionally, by culturing paired and unpaired SMCs on microfabricated adhesive islands of different areas, we found that N-cadherin relaxes the spreading requirement for SMC proliferation. In vivo SMC deletion of N-cadherin strongly reduced injury-induced cycling. Finally, SMC-specific deletion of FAK inhibited proliferation after vascular injury, and this was accompanied by reduced induction of N-cadherin. Thus, a stiffness- and FAK-dependent induction of N-cadherin connects cell-matrix to cell-cell adhesion and regulates the degree of cell spreading needed for cycling.

A FAK-Cas-Rac-lamellipodin signaling module transduces extracellular matrix stiffness into mechanosensitive cell cycling.

Tissue and extracellular matrix (ECM) stiffness is transduced into intracellular stiffness, signaling, and changes in cellular behavior. Integrins and several of their associated focal adhesion proteins have been implicated in sensing ECM stiffness. We investigated how an initial sensing event is translated into intracellular stiffness and a biologically interpretable signal. We found that a pathway consisting of focal adhesion kinase (FAK), the adaptor protein p130Cas (Cas), and the guanosine triphosphatase Rac selectively transduced ECM stiffness into stable intracellular stiffness, increased the abundance of the cell cycle protein cyclin D1, and promoted S-phase entry. Rac-dependent intracellular stiffening involved its binding partner lamellipodin, a protein that transmits Rac signals to the cytoskeleton during cell migration. Our findings establish that mechanotransduction by a FAK-Cas-Rac-lamellipodin signaling module converts the external information encoded by ECM stiffness into stable intracellular stiffness and mechanosensitive cell cycling. Thus, lamellipodin is important not only in controlling cellular migration but also for regulating the cell cycle in response to mechanical signals.

The effects of 17ß-estradiol and a selective estrogen receptor modulator, bazedoxifene, on ovarian carcinogenesis.

OBJECTIVE: To test if estrogen promotes carcinogenesis in vitro and in a genetic mouse model of ovarian cancer and whether its effects can be inhibited by a novel selective estrogen receptor modulator (SERM), bazedoxifene. METHODS: Bazedoxifene was synthesized and it was confirmed that the drug abrogated the uterine stimulatory effect of 17ß-estradiol in mice. To determine if hormones alter tumorigenesis in vivo LSL-K-ras(G12D/+)Pten(loxP/loxP) mice were treated with vehicle control, 17ß-estradiol or bazedoxifene. Hormone receptor status of a cell line established from LSL-K-ras(G12D/+)Pten(loxP/loxP) mouse ovarian tumors was characterized using Western blotting and immunohistochemistry. The cell line was treated with hormones and invasion assays were performed using Boyden chambers and proliferation was assessed using MTT assays. RESULTS: In vitro 17ß-estradiol increased both the invasion and proliferation of ovarian cancer cells and bazedoxifene reversed these effects. However, in the genetic mouse model neither treatment with 17ß-estradiol nor bazedoxifene changed mean tumor burden when compared to treatment with placebo. The mice in all treatment groups had similar tumor incidence, metastatic nodules and ascites. CONCLUSION: While 17ß-estradiol increases the invasion and proliferation of ovarian cancer cells, these effects do not translate into increased tumor burden in a genetic mouse model of endometrioid ovarian cancer. Likewise, while the SERM reversed the detrimental effects of estrogen in vitro, there was no change in tumor burden in mice treated with bazedoxifene. These findings demonstrate the complex interplay between hormones and ovarian carcinogenesis.

Effects of oral contraceptives or a gonadotropin-releasing hormone agonist on ovarian carcinogenesis in genetically engineered mice.

Although epidemiologic evidence for the ability of combined oral contraception (OC) to reduce the risk of ovarian cancer (OvCa) is convincing, the biological mechanisms underlying this effect are largely unknown. We conducted the present study to determine if OC also influences ovarian carcinogenesis in a genetic mouse model and, if so, to investigate the mechanism underlying the protective effect. LSL-K-ras(G12D/+)Pten(loxP/loxP) mice were treated with ethinyl estradiol plus norethindrone, contraceptive hormones commonly used in combined OC, or norethindrone alone, or a gonadotropin-releasing hormone agonist. The combined OC had a 29% reduction in mean total tumor weight compared with placebo (epithelial tumor weight, -80%). Norethindrone alone reduced mean total tumor weight by 42% (epithelial tumor weight, -46%), and the gonadotropin-releasing hormone agonist increased mean total tumor weight by 71% (epithelial tumor weight, +150%). Large variations in tumor size affected the P values for these changes, which were not statistically significant. Nonetheless, the OC reductions are consistent with the epidemiologic data indicating a protective effect of OC. Matrix metalloproteinase-2 activity was decreased in association with OC, indicating that OC may affect ovarian carcinogenesis by decreasing proteolytic activity, an important early event in the pathogenesis of OvCa. In contrast, OC increased invasion in a K-ras/Pten OvCa cell line established from the mouse tumors, suggesting that OC hormones, particularly estrogen, may have a detrimental effect after the disease process is under way. Our study results support further investigation of OC effects and mechanisms for OvCa prevention.

Regulation of gonadotropin-releasing hormone in nonhypothalamic tissues.

It is well established that hypothalamic gonadotropin-releasing hormone (GnRH) controls reproductive function by stimulating the production of gonadotropins from the pituitary. GNRH gene and its receptor (GnRHR) have also been detected outside the hypothalamus, and a growing body of literature supports an extrapituitary role for GnRH action. The exact function of GnRH in these tissues is not known, but GnRH expression has been described in reproductive tissues, including the ovary, placenta, breast, testes, and prostate. This article provides an overview of the regulation of GnRH gene expression in nonhypothalamic reproductive tissues. After GnRH gene structure is reviewed, the physiologic role of GnRH and regulation of its expression in several reproductive tissues are examined. When possible, transcriptional regulation is discussed, but due to low levels of expression, transcriptional regulation of GnRH in extrahypothalamic tissues has been extremely difficult to study. Consequently, the factors that mediate GnRH gene expression in these tissues are only beginning to be identified.