Reversible epithelial-to-mesenchymal transition (EMT) is central to tissue development, epithelial stemness, and cancer metastasis. While many regulatory elements have been identified to induce EMT, the complex process underlying such cellular plasticity remains poorly understood. Utilizing a systems biology approach integrating modeling and experiments, we found multiple intermediate states contributing to EMT and that the robustness of the transitions is modulated by transcriptional factor Ovol2. In particular, we obtained evidence for a mutual inhibition relationship between Ovol2 and EMT inducer Zeb1, and observed that adding this regulation generates a novel four-state system consisting of two distinct intermediate phenotypes that differ in differentiation propensities and are favored in different environmental conditions. We identified epithelial cells that naturally exist in an intermediate state with bidirectional differentiation potential, and found the balance between EMT-promoting and -inhibiting factors to be critical in achieving and selecting between intermediate states. Our analysis suggests a new design principle in controlling cellular plasticity through multiple intermediate cell fates and underscores the critical involvement of Ovol2 and its associated molecular regulations.

SUMMARY Epithelial cells possess remarkable plasticity, having the ability to become mesenchymal cells through alterations in adhesion and motility (epithelial-to-mesenchymal transition [EMT]). However, how epithelial plasticity is kept in check in epithelial cells during tissue development and regeneration remains to be fully understood. Here we show that restricting the EMT of mammary epithelial cells by transcription factor Ovol2 is required for proper morphogenesis and regeneration. Deletion of Ovol2 blocks mammary ductal morphogenesis, depletes stem and progenitor cell reservoirs, and leads epithelial cells to undergo EMT in vivo to become nonepithelial cell types. Ovol2 directly represses myriad EMT inducers, and its absence switches response to TGF-β from growth arrest to EMT. Furthermore, forced expression of the repressor isoform of Ovol2 is able to reprogram metastatic breast cancer cells from a mesenchymal to an epithelial state. Our findings underscore the critical importance of exquisitely regulating epithelial plasticity in development and cancer.

During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/2-deficient epidermal cells fail to undertake α-catenin–driven actin cytoskeletal reorganization and adhesive maturation, and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations as well as defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-α-catenin sequential repression and highlight functions of Ovol as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity.

Mesenchymal stem cells (MSCs) are known to both have powerful immunosuppressive properties and promote allograft tolerance. Determining the environmental oxygen tension and inflammatory conditions under which MSCs are optimally primed for this immunosuppressive function is essential to their utilization in promoting graft tolerance. Of particular interest is the mechanisms governing the interaction between MSCs and regulatory T cells (Tregs), which is relatively unknown. We performed our experiments utilizing rat bone marrow derived MSCs. We observed that priming MSCs in hypoxia promotes maintenance of stem-like characteristics, with greater expression of typical MSC cell-surface markers, increased proliferation, and maintenance of differentiation potential. Addition of autologous MSCs to CD4+/allogeneic endothelial cell (EC) co-culture increases regulatory T cell (Treg) proliferation, which is further enhanced when MSCs are primed in hypoxia. Furthermore, MSC-mediated Treg expansion does not require direct cell-cell contact. The expression of indolamine 2,3-dioxygenase, a mediator of MSC immunomodulation, increases when MSCs are primed in hypoxia, and inhibition of IDO significantly decreases the expansion of Tregs. Priming with inflammatory cytokines IFNγ and TNFα increases also expression of markers associated with MSC immunomodulatory function, but decreases MSC proliferation. The expression of IDO also increases when MSCs are primed with inflammatory cytokines. However, there is no increase in Treg expansion when MSCs are primed with IFNγ, suggesting an alternate mechanism for inflammatory-stimulated MSC immunomodulation. Overall, these results suggest that MSCs primed in hypoxia or inflammatory conditions are optimally primed for immunosuppressive function. These results provide a clearer picture of how to enhance MSC immunomodulation for clinical use.

Highlights d Epidermal Nrf2 initiates a regenerative response through Ccl2 regulation d Ccl2 release by basal stem/progenitor keratinocytes prompts macrophage trafficking d Ccl2 regulates EGF production in macrophages trafficked to the injury site d EGF from macrophages stimulates basal keratinocyte proliferation

Highlights d Maintenance of mammary basal cell fate and stem cell quiescence requires Zeb1 d Zeb1 promotes basal cell fate in part through EMTassociated gene regulation d Zeb1 acts in quiescent basal cells to promote self-renewal and suppress proliferation d Zeb1 suppression of Wnt signaling is important for basal stem cell maintenance

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Purpose: Despite advancements in disease management and wound therapies, chronic diabetic ulcers represent the leading cause of non-traumatic lower extremity amputations in the United States. The Nrf2 master antioxidant transcription factor has emerged as a promising target for combatting the oxidative stress underlying these wounds. We have previously implicated Keap1-mediated Nrf2 dysfunction in delayed diabetic wound healing, and as a therapeutic target, in mice. However, whether these findings extend to human diabetic wounds remains unknown. Methods: Intact skin (S) and wound tissue (W) was obtained from diabetic (D) and nondiabetic (ND) patients, and analyzed for reactive oxygen species (ROS) end products (8-OhDG DNA assay), and with immunohistochemistry (IHC), qPCR (gene expression), and western blot (protein expression). Student’s t-tests were used for statistical analysis. Results: Both DS and DW contained more than 2-fold ROS end products, respectively, compared to NDS and NDW (2.344 vs. 1.117 ng/mL and 4.648 vs. 1.866 ng/mL, p<0.008). DS and DW exhibited 2-fold and 2.5-fold decreases in epidermal Nrf2 staining, respectively, compared to NS and NW (p<0.001). Keap1-the cytoplasmic Nrf2 repressor-was overexpressed in DW when compared to NDW (p<0.04). DW showed a 75% reduction in nuclear Nrf2 protein expression (p<0.02) as compared to NDW. DW also exhibited a 25% relative reduction in gene expression of MnSOD, a key Nrf2 downstream antioxidant enzyme, and 5-fold decrease in epidermal cytoplasmic MnSOD staining compared to NDW, p<0.03. Conclusion: Here, we implicate Nrf2 dysfunction in impaired redox homeostasis and consequent oxidative damage in both human diabetic skin and diabetic wounds as compared to nondiabetics. We also identify Keap1 as a likely mediator of impaired healing in diabetic wounds. These results recapitulate our previous findings in mice, further validating this critical pathway as a promising target for understanding and treating diabetic wound healing. Disclosure J.A. David: None. A.P. Villarreal-Ponce: None. S.A. Abdou: None. D.L. Sultan: None. W.J. Rifkin: None. J. Kwong: None. P.S. Rabbani: None. D. Ceradini: None.