Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization.

Epithelial-mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis.

Heterogeneity of myotubes generated by the MyoD and E12 basic helix-loop-helix transcription factors in otherwise non-differentiation growth conditions.

We used a synthetic biology approach to produce myotubes from mammalian C2C12 myoblasts in non-differentiation growth conditions using the expression of basic helix-loop-helix transcription factors, MyoD and E12, in various combinations and configurations. Our approach not only recapitulated the basics of muscle development and physiology, as the obtained myotubes showed qualities similar to those seen in striated muscle fibers in vivo, but also allowed for the synthesis of populations of myotubes which assumed distinct morphology, myofibrillar development and Ca(2+) dynamics. This fashioned class of biomaterials is suitable for the building blocks of soft actuators in micro-scale biomimetic robotics. This production line strategy can be embraced in reparative medicine as synthetic human myotubes with predetermined morphological/functional properties could be obtained using this very approach. This methodology can be adopted beyond striated muscle for the engineering of other tissue components/cells whose differentiation is governed by the principles of basic helix-loop-helix transcription factors, as in the case, for example, of neural or immune cell types.

E47 and Id1 interplay in epithelial-mesenchymal transition.

E12/E47 proteins (encoded by E2A gene) are members of the class I basic helix-loop-helix (bHLH) transcription factors (also known as E proteins). E47 has been described as repressor of E-cadherin and inducer of epithelial-mesenchymal transition (EMT). We reported previously that EMT mediated by E47 in MDCK cells occurs with a concomitant overexpression of Id1 and Id3 proteins. Id proteins belong to class V of HLH factors that lack the basic domain; they dimerise with E proteins and prevent their DNA interaction, thus, acting as dominant negative of E proteins. Here, we show that E47 interacts with Id1 in E47 overexpressing MDCK cells that underwent a full EMT as well as in mesenchymal breast carcinoma and melanoma cell lines. By conducting chromatin immunoprecipitation assays we demonstrate that E47 binds directly to the endogenous E-cadherin promoter of mesenchymal MDCK-E47 cells in a complex devoid of Id1. Importantly, our data suggest that both E47 and Id1 are required to maintain the mesenchymal phenotype of MDCK-E47 cells. These data support the collaboration between E47 and Id1 in the maintenance of EMT by mechanisms independent of the dominant negative action of Id1 on E47 binding to E-cadherin promoter. Finally, the analysis of several N0 breast tumour series indicates that the expression of E47 and ID1 is significantly associated with the basal-like phenotype supporting the biological significance of the present findings.

Essential role of AWP1 in neural crest specification in Xenopus.

The neural crest (NC) comprises a transient and multipotent embryonic cell population, which gives rise to a wide variety of cell types, including craniofacial cartilage, melanocytes, and neurons and glia of the peripheral nervous system. The NC is induced by the integrated action of Wnt, FGF, and BMP signaling, and its cell fates are subsequently specified by a genetic cascade of specific transcription factors. Here we describe a critical role of AWP1 in NC induction during Xenopus early development. Xenopus AWP1 (XAWP1) was found to be expressed in the presumptive preplacodal ectoderm, neural tissue, and posterior dorsal mesoderm, but was absent in the neural fold along the anterior-posterior axis of the neurulae. Notably, XAWP1 was induced by FGF8a in naïve ectodermal tissue. XAWP1-depleted embryos exhibited defects in pigmentation, craniofacial cartilage, and in the dorsal fin. A knockdown of XAWP1 impaired both endogenous and the FGF8a or Wnt8-induced expression of NC markers without affecting mesoderm formation. Furthermore, NC induction inhibited by XAWP1 depletion was rescued by co-expression of activating forms of beta-catenin or TCF3. In addition, overexpression of XAWP1, in concert with BMP inhibition, induced the expression of neural plate border specifiers, Pax3 and Msx1, and these regulatory factors recovered NC induction in the XAWP1-depleted embryos. Beta-catenin stability and Wnt-responsive reporter activity were also impaired in AWP1-depleted cells. Taken together, these results suggest that XAWP1 functions as a mediator of Wnt signaling to regulate NC specification.

E proteins regulate osteoclast maturation and survival.

Osteoclasts are bone-specific polykaryons derived from myeloid precursors under the stimulation of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL). E proteins are basic helix-loop-helix (bHLH) transcription factors that modulate lymphoid versus myeloid cell fate decisions. To study the role of E proteins in osteoclasts, myeloid-specific E protein gain-of-function transgenic mice were generated. These mice have high bone mass due to decreased osteoclast numbers and increased osteoclast apoptosis leading to overall reductions in resorptive capacity. The molecular mechanism of decreased osteoclast numbers and resorption is in part a result of elevated expression of CD38, a regulator of intracellular calcium pools with known antiosteoclastogenic properties, which increases sensitivity to apoptosis. In vivo, exogenous RANKL stimulation can overcome this inhibition to drive osteoclastogenesis and bone loss. In vitro-derived ET2 osteoclasts are more spread and more numerous with increases in RANK, triggering receptor expressed on myeloid cells 2 (TREM2), and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) compared to wild type. However, their resorptive capacity does not increase accordingly. Thus, E proteins participate in osteoclast maturation and survival in homeostatic bone remodeling.

Snail1, Snail2, and E47 promote mammary epithelial branching morphogenesis.

Several E-box-binding transcription factors regulate individual and collective cell migration and enhance the motility of epithelial cells by promoting epithelial-mesenchymal transition (EMT). Here, we characterized the role of a subset of these transcription factors and the EMT proteome in branching morphogenesis of mammary epithelial tissues using a three-dimensional organotypic culture model of the mammary duct. We found that the transcription factors Snail1, Snail2, and E47 were transiently upregulated at branch sites; decreasing the expression of these transcription factors inhibited branching. Conversely, ectopic expression of Snail1, Snail2, and E47 induced branching in the absence of exogenous stimuli. These changes correlated with the expression of mesenchymal markers and repression of E-cadherin, which was essential for branching. Snail1 and Snail2 also promoted cell survival at branch sites, but this was not sufficient to induce branching. These findings indicate that Snail1, Snail2, and E47 can promote collective migration during branching morphogenesis of mammary epithelial tissues through key regulators of EMT.

E47 regulates hematopoietic stem cell proliferation and energetics but not myeloid lineage restriction.

The immune system is replenished by self-renewing hematopoietic stem cells (HSCs) that produce multipotent progenitors (MPPs) with little renewal capacity. E-proteins, the widely expressed basic helix-loop-helix transcription factors, contribute to HSC and MPP activity, but their specific functions remain undefined. Using quantitative in vivo and in vitro approaches, we show that E47 is dispensable for the short-term myeloid differentiation of HSCs but regulates their long-term capabilities. E47-deficient progenitors show competent myeloid production in short-term assays in vitro and in vivo. However, long-term myeloid and lymphoid differentiation is compromised because of a progressive loss of HSC self-renewal that is associated with diminished p21 expression and hyperproliferation. The activity of E47 is shown to be cell-intrinsic. Moreover, E47-deficient HSCs and MPPs have altered expression of genes associated with cellular energy metabolism, and the size of the MPP pool but not downstream lymphoid precursors in bone marrow or thymus is rescued in vivo by antioxidant. Together, these observations suggest a role for E47 in the tight control of HSC proliferation and energy metabolism, and demonstrate that E47 is not required for short-term myeloid differentiation.