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Wound healing is a highly conserved process that restores the integrity and functionality of injured tissues. Transforming growth factor (TGF)-β is a master regulator of wound healing, whose signaling is attenuated by the E3 ubiquitin ligase Smurf2. Herein, the roles of Smurf2 in cutaneous wound healing were examined using a murine incisional cutaneous model. Loss of Smurf2increased early inflammation in the wounds and led to narrower wounds with greater breaking strength. Loss of Smurf2also led to more linearized collagen bundles in normal and wounded skin. Gene expression analyses by real-time quantitative PCR indicated that Smurf2-deficient fibroblasts had increased levels of TGF-β/Smad3 signaling and changes in expression profile of genes related to matrix turnover. The effect of Smurf2loss on wound healing and collagen bundling was attenuated by the heterozygous loss of Smad3. Together, these results show that Smurf2 affects inflammation and collagen processing in cutaneous wounds by down-regulating TGF-β/Smad3 signaling.

Transforming growth factor-β (TGF-β) is major inducer of epithelial-to-mesenchymal transition (EMT), which associates with cancer cell metastasis and resistance to chemotherapy and targeted drugs, through both transcriptional and non-transcriptional mechanisms. We previously reported that, in cancer cells, heightened mitogenic signaling allows TGF-β-activated Smad3 to interact with poly(RC) binding protein 1 (PCBP1) and together they regulate many alternative splicing events that favors expression of protein isoforms essential for EMT, cytoskeletal rearrangement, and adherens junction signaling. Here we show that the exclusion of TGF-β-activated kinase 1 (TAK1) variable exon 12 requires another RNA-binding protein, Fox-1 homolog 2 (Rbfox2), which binds intronic sequences in front of exon 12 independently of the Smad3–PCBP1 complex. Functionally, exon 12-excluded TAK1∆E12 and full-length TAK1FL are distinct. The short isoform TAK1∆E12 is constitutively active and supports TGF-β-induced EMT and nuclear factor kappa B (NF-κB) signaling, whereas the full-length isoform TAK1FL promotes TGF-β-induced apoptosis. These observations offer a harmonious explanation for how a single TAK1 kinase can mediate the opposing responses of cell survival and apoptosis in response to TGF-β. They also reveal a propensity of the alternatively spliced TAK1 isoform TAK1∆E12 to cause drug resistance due to its activity in supporting EMT and NF-κB survival signaling.

Metastasis remains the greatest challenge in the clinical management of cancer. Cell motility is a fundamental and ancient cellular behaviour that contributes to metastasis and is conserved in simple organisms. In this Review, we evaluate insights relevant to human cancer that are derived from the study of cell motility in non-mammalian model organisms. Dictyostelium discoideum, Caenorhabditis elegans, Drosophila melanogaster and Danio rerio permit direct observation of cells moving in complex native environments and lend themselves to large-scale genetic and pharmacological screening. We highlight insights derived from each of these organisms, including the detailed signalling network that governs chemotaxis towards chemokines; a novel mechanism of basement membrane invasion; the positive role of E-cadherin in collective direction-sensing; the identification and optimization of kinase inhibitors for metastatic thyroid cancer on the basis of work in flies; and the value of zebrafish for live imaging, especially of vascular remodelling and interactions between tumour cells and host tissues. While the motility of tumour cells and certain host cells promotes metastatic spread, the motility of tumour-reactive T cells likely increases their antitumour effects. Therefore, it is important to elucidate the mechanisms underlying all types of cell motility, with the ultimate goal of identifying combination therapies that will increase the motility of beneficial cells and block the spread of harmful cells.

Epithelial cells in the skin and other tissues rely on signals from their environment to maintain homeostasis and respond to injury, and GPCRs play a critical role in this communication. A better understanding of the GPCRs expressed in epithelial cells will contribute to understanding the relationship between cells and their niche and could lead to developing new therapies to modulate cell fate. This study used human primary keratinocytes as a model to investigate the specific GPCRs regulating epithelial cell proliferation and differentiation. We identified 3 key receptors—HCAR3, LTB4R, and GPR137—and found that knockdown of these receptors led to changes in numerous gene networks that are important for maintaining cell identity and promoting proliferation while inhibiting differentiation. Our study also revealed that the metabolite receptor HCAR3 regulates keratinocyte migration and cellular metabolism. Knockdown of HCAR3 led to reduced keratinocyte migration and respiration, which could be attributed to altered metabolite use and aberrant mitochondrial morphology caused by the absence of the receptor. This study contributes to understanding the complex interplay between GPCR signaling and epithelial cell fate decisions.

Canonical TGF-β is involved in cell differentiation, tissue maintenance, and wound healing, but also plays a central role in the pathogenesis of diseases such as cancer. Here we describe a lentivirus-based reporter vector system expressing green fluorescent protein (GFP) or red fluorescent protein (RFP) under the control of a Smad3-responsive element (CAGA)12that allows observation of the temporospatial pattern of endogeneous Smad3-mediated signaling on a cellular level. Use of this method will be valuable to identify cells with active Smad3 signaling and investigate the role of endogenous Smad3 signaling in complex systems such as co-cultures in vitro, or in tumors during tumor cell invasion and metastasis in vivo.

Comment on: Aprelikova O, et al. Cell Cycle 2010; 9:4387–98.

Introduction: Molecular dissection of the signaling pathways that underlie complex biological responses in the mammary epithelium is limited by the difficulty of propagating large numbers of mouse mammary epithelial cells, and by the inability of ribonucleic acid interference-based knockdown approaches to fully ablate gene function. Here we describe a method for the generation of conditionally immortalized mammary epithelial cells with defined genetic defects, and we show how such cells can be used to investigate complex signal transduction processes using the transforming growth factor beta (TGFß)/Smad pathway as an example. Methods: We intercrossed the previously described H-2Kb-tsA58 transgenic mouse (Immortomouse), which expresses a temperature-sensitive mutant of the simian virus-40 large T-antigen (tsTAg), with mice of differing Smad genotypes. Conditionally immortalized mammary epithelial cell cultures were derived from the virgin mammary glands of offspring of these crosses and were used to assess the Smad dependency of different biological responses to TGFß. Results: IMECs could be propagated indefinitely at permissive temperatures and had a stable epithelial phenotype, resembling primary mammary epithelial cells with respect to several criteria, including responsiveness to TGFß. Using this panel of cells, we demonstrated that Smad3, but not Smad2, is necessary for TGFß-induced apoptotic, growth inhibitory and epithelial-to-mesenchymal transition responses, whereas either Smad2 or Smad3 can support TGFß-induced invasion as long as a threshold level of total Smad is exceeded. Conclusions: The present work demonstrates the practicality and utility of generating conditionally immortalized mammary epithelial cell lines from genetically modified Immortomice for detailed investigation of complex signaling pathways in the mammary epithelium.