β1 Integrin Is Essential for Teratoma Growth and Angiogenesis

Teratomas are benign tumors that form after ectopic injection of embryonic stem (ES) cells into mice and contain derivatives of all primitive germ layers. To study the role of β1 integrin during teratoma formation, we compared teratomas induced by normal and β1-null ES cells. Injection of normal ES cells gave rise to large teratomas. In contrast, β1-null ES cells either did not grow or formed small teratomas with an average weight of <5% of that of normal teratomas. Histological analysis of β1-null teratomas revealed the presence of various differentiated cells, however, a much lower number of host-derived stromal cells than in normal teratomas. Fibronectin, collagen I, and nidogen were expressed but, in contrast to normal teratomas, diffusely deposited in β1-null teratomas. Basement membranes were present but with irregular shape and detached from the cell surface

MRCKα Is Dispensable for Breast Cancer Development in the MMTV-PyMT Model.

MRCKα is a ubiquitously expressed serine/threonine kinase involved in cell contraction and F-actin turnover, which is highly amplified in human breast cancer and part of a gene expression signature for bad prognosis. Nothing is known about the in vivo function of MRCKα. To explore MRCKα function in development and in breast cancer, we generated mice lacking a functional MRCKα gene. Mice were born close to the Mendelian ratio and showed no obvious phenotype including a normal mammary gland formation. Assessing breast cancer development using the transgenic MMTV-PyMT mouse model, loss of MRCKα did not affect tumor onset, tumor growth and metastasis formation. Deleting MRCKα and its related family member MRCKβ in two triple-negative breast cancer cell lines resulted in reduced invasion of MDA-MB-231 cells, but did not affect migration of 4T1 cells. Further genomic analysis of human breast cancers revealed that MRCKα is frequently co-amplified with the oncogenes ARID4B and AKT3 which might contribute to the prognostic value of MRCKα expression. Collectively, these data suggest that MRCKα might be a prognostic marker for breast cancer, but probably of limited functional importance

β1 Integrin-Mediated Adhesion Signalling Is Essential for Epidermal Progenitor Cell Expansion

Background: There is a major discrepancy between the in vitro and in vivo results regarding the role of b1 integrins in the maintenance of epidermal stem/progenitor cells. Studies of mice with skin-specific ablation of b1 integrins suggested that epidermis can form and be maintained in their absence, while in vitro data have shown a fundamental role for these adhesion receptors in stem/progenitor cell expansion and differentiation. Methodology/Principal Findings: To elucidate this discrepancy we generated hypomorphic mice expressing reduced b1 integrin levels on keratinocytes that developed similar, but less severe defects than mice with b1-deficient keratinocytes. Surprisingly we found that upon aging these abnormalities attenuated due to a rapid expansion of cells, which escaped or compensated for the down-regulation of b1 integrin expression. A similar phenomenon was observed in aged mice with a complete, skin-specific ablation of the b1 integrin gene, where cells that escaped Cre-mediated recombination repopulated the mutant skin in a very short time period. The expansion of b1 integrin expressing keratinocytes was even further accelerated in situations of increased keratinocyte proliferation such as wound healing. Conclusions/Significance: These data demonstrate that expression of b1 integrins is critically important for the expansion of epidermal progenitor cells to maintain epidermal homeostasis

Microglia dysfunction caused by the loss of Rhoa disrupts neuronal physiology and leads to neurodegeneration

© 2020 The Author(s). Creative Commons Attribution (CC BY 4.0)Nervous tissue homeostasis requires the regulation of microglia activity. Using conditional gene targeting in mice, we demonstrate that genetic ablation of the small GTPase Rhoa in adult microglia is sufficient to trigger spontaneous microglia activation, producing a neurological phenotype (including synapse and neuron loss, impairment of long-term potentiation [LTP], formation of β-amyloid plaques, and memory deficits). Mechanistically, loss of Rhoa in microglia triggers Src activation and Src-mediated tumor necrosis factor (TNF) production, leading to excitotoxic glutamate secretion. Inhibiting Src in microglia Rhoa-deficient mice attenuates microglia dysregulation and the ensuing neurological phenotype. We also find that the Rhoa/Src signaling pathway is disrupted in microglia of the APP/PS1 mouse model of Alzheimer disease and that low doses of Aβ oligomers trigger microglia neurotoxic polarization through the disruption of Rhoa-to-Src signaling. Overall, our results indicate that disturbing Rho GTPase signaling in microglia can directly cause neurodegeneration.The authors acknowledge the support of the following i3S Scientific Platforms: Animal Facility, Translational Cytometry Unit (TraCy), BioSciences Screening (BS) and Advanced Light Microscopy (ALM), and members of the national infrastructure PPBI-Portuguese Platform of BioImaging (supported by POCI-01–0145-FEDER-022122). FCT Portugal ( PTDC/MED-NEU/31318/2017-031318 ) supported work in the J.B.R. lab. FCT Portugal , PEst ( UID/NEU/04539/2013 ), COMPETE-FEDER ( POCI-01-0145-FEDER-007440 ), Centro 2020 Regional Operational Programme ( CENTRO-01-0145-FEDER-000008 : BrainHealth 2020), and Strategic Project UIDB/04539/2020 and UIDP/04539/2020 (CIBB) supported work in the A.F.A. lab. C.C.P. and R.S. hold employment contracts financed by national funds through FCT (Fundação para a Ciência e a Tecnologia, I.P.) in the context of the program contract described in paragraphs 4, 5, and 6 of article 23 of law no. 57/2016, of August 29th, as amended by law no. 57/2017 of July 19th.info:eu-repo/semantics/publishedVersio

Coordination by Cdc42 of actin, contractility, and adhesion for melanoblast movement in mouse skin

YesThe individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics.Cancer Research UK (to L.M.M. [A17196], R.H.I. [A19257], and S.W.G.T.) and NIH grants P01-GM103723 and P41-EB002025 (to K.M.H.). N.R.P. is supported by a Pancreatic Cancer Research Fund grant (to L.M.M.). Funding to Prof. Rottner by the Deutsche Forschungsgemeinschaft (grant RO2414/3-2)

Maintaining resting cardiac fibroblasts in vitro by disrupting mechanotransduction

Mechanical cues activate cardiac fibroblasts and induce differentiation into myofibroblasts, which are key steps for development of cardiac fibrosis. In vitro, the high stiffness of plastic culturing conditions will also induce these changes. It is therefore challenging to study resting cardiac fibroblasts and their activation in vitro. Here we investigate the extent to which disrupting mechanotransduction by culturing cardiac fibroblasts on soft hydrogels or in the presence of biochemical inhibitors can be used to maintain resting cardiac fibroblasts in vitro. Primary cardiac fibroblasts were isolated from adult mice and cultured on plastic or soft (4.5 kPa) polyacrylamide hydrogels. Myofibroblast marker gene expression and smooth muscle α-actin (SMA) fibers were quantified by real-time PCR and immunostaining, respectively. Myofibroblast differentiation was prevented on soft hydrogels for 9 days, but had occurred after 15 days on hydrogels. Transferring myofibroblasts to soft hydrogels reduced expression of myofibroblast-associated genes, albeit SMA fibers remained present. Inhibitors of transforming growth factor β receptor I (TGFβRI) and Rho-associated protein kinase (ROCK) were effective in preventing and reversing myofibroblast gene expression. SMA fibers were also reduced by blocker treatment although cell morphology did not change. Reversed cardiac fibroblasts maintained the ability to re-differentiate after the removal of blockers, suggesting that these are functionally similar to resting cardiac fibroblasts. However, actin alpha 2 smooth muscle (Acta2), lysyl oxidase (Lox) and periostin (Postn) were no longer sensitive to substrate stiffness, suggesting that transient treatment with mechanotransduction inhibitors changes the mechanosensitivity of some fibrosis-related genes. In summary, our results bring novel insight regarding the relative importance of specific mechanical signaling pathways in regulating different myofibroblast-associated genes. Furthermore, combining blocker treatment with the use of soft hydrogels has not been tested previously and revealed that only some genes remain mechano-sensitive after phenotypic reversion. This is important information for researchers using inhibitors to maintain a "resting" cardiac fibroblast phenotype in vitro as well as for our current understanding of mechanosensitive gene regulation