The CASZ1/Egfl7 transcriptional pathway is required for RhoA expression in vascular endothelial cells

Abstract

Vertebrate development depends on the formation of a closed circulatory loop consisting of intricate networks of veins, arteries, and lymphatic vessels. The coordinated participation of multiple molecules including growth factors, transcription factors, extracellular matrix proteins, and regulators of signaling such as small GTPases is essential for eliciting the desired cellular behaviors associated with vascular assembly and morphogenesis. We have recently demonstrated that a novel transcriptional pathway involving activation of the Epidermal Growth Factor-like Domain 7 (Egfl7) gene by the transcription factor CASTOR (CASZ1) is required for blood vessel assembly and lumen morphogenesis. Furthermore, this transcriptional network promotes RhoA expression and subsequent GTPase activity linking transcriptional regulation of endothelial gene expression to direct physiological outputs associated with Rho GTPase signaling, i.e., cell adhesion and cytoskeletal dynamics. Here we will discuss our studies with respect to our current understanding of the mechanisms underlying regulation of RhoA transcription, protein synthesis, and activity.

The cardiovascular system is the first organ system to develop and function during embryonic development. Proper establishment of the circulatory system is essential for meeting the demands of a rapidly growing embryo in need of nutrients, gas exchange, and waste removal. Blood vessel development occurs via vasculogenesis and angiogenesis. During vasculogenesis, endothelial cells, the building blocks of blood vessels, are first specified from the mesoderm whereby they subsequently migrate to precise locations within the embryo to assemble into primitive vascular structures.^1,2 From this point on, most of vascular development proceeds via angiogenesis in which vessels branch or sprout from pre-existing vessels ultimately giving rise to a specialized hierarchy of veins, arteries, and lymphatic vessels.^3,4 The coordination of endothelial cell behaviors including migration, proliferation, and adhesive properties between cells and between the underlying extracellular matrix (ECM) is critical for these key steps of vascular assembly, and hence dysregulation of these behaviors can lead to a number of disease states such as atherosclerosis, stroke, rheumatoid arthritis, and tumorigenesis.^5,6 An understanding of the molecular and cellular mechanisms underlying endothelial cell behavior is therefore necessary to provide a basis for the design of therapeutics.

Endothelial cells are highly sensitive to influences from the environment and alter their behaviors in response to signaling cascades typically initiated extracellularly through cell surface receptors. One key family of proteins known to play a critical role in signal transduction is the Rho family of small GTPases. The Rho GTPases, including members of the Rho, Rac, and Cdc42 sub-families, are critical regulators of the actin cytoskeleton and promote the formation of distinct actin bundles such as actin stress fibers, lamellipodia, or filopodia respectively.⁷ Like all GTPases, RhoA is regulated by GTP binding and cycles between the active GTP-bound form and the inactive GDP-bound form. RhoA activity is tightly controlled by Guanine nucleotide Exchange Factors (GEFs) that facilitate exchange of GDP for GTP, GTPase Activating Proteins (GAPs) that facilitate RhoA’s intrinsic GTPase activity (to inhibit RhoA), and RhoGDIs which sequester RhoA into an inactive sub-cellular fraction. In its GTP-bound form, RhoA interacts with a variety of effector molecules that mediate its effects on the actin cytoskeleton including the Rho-kinases (ROCK 1 and II), diaphanous-related formins (mDia1 and mDia2), protein kinase N, citron kinase, rhophilin, and Rhotekin. The mDia proteins directly catalyze actin polymerization in cooperation with the actin binding protein, profilin; while ROCK stimulates actin polymerization by inhibiting the disassembly of actin polymers through LIM-kinase-dependent inhibition of cofilin. ROCK also inhibits myosin phosphatase to stimulate actin-myosin based contraction, which promotes actin bundling and stress fiber formation.⁷

Recent studies indicate that besides regulating cell shape changes, Rho-dependent signals can directly impact gene expression. Indeed, important SRF-cofactors including Myocardin-related transcription factors (i.e., MRTF-A and MRTF-B) have been shown to contain monomeric (G)-actin binding domains, and association with G-actin masks a nuclear localization targeting sequence resulting in their cytoplasmic sequestration. Thus, upon RhoA-induced G-actin polymerization, the fall in free cytoplasmic G-actin results in G-actin release from MRTFs therefore promoting nuclear accumulation and facilitating MRTF-dependent gene transcription.^8,9 The Rho/MRTF/SRF pathway is a key means by which the smooth muscle cell phenotype is regulated¹⁰ and recent studies indicate that it may also be particularly important for determining endothelial cell fates as perturbation of the Rho-MRTF axis impairs endothelial cell differentiation from mesenchymal stem cells and conversely, the endothelial to mesenchymal transition.^11,12 RhoA-mediated signaling has also been implicated in NF-κB-dependent gene transcription in various cell types including endothelial cells thereby promoting angiogenesis, at least in part, by upregulating VEGFR2 levels.¹³

Accumulating evidence has highlighted significant roles for RhoA in vascular biology including angiogenesis, lumen formation, and endothelial barrier function.^14-16

We have recently further implicated RhoA-mediated signaling in vascular development by showing that RhoA lies downstream of a transcriptional cascade involving the transcription factor CASTOR (CASZ1) and its direct target Epidermal Growth Factor-like Domain 7 (Egfl7).^17,18 CASZ1 is an evolutionarily conserved transcription factor originally characterized in Drosophila to maintain neural stem cell identity.^19,20 In addition to expression domains in the brain, Casz1 transcripts have been identified in cardiovascular tissues across vertebrate species, and work from our lab demonstrated that CASZ1 is required for Xenopus cardiogenesis by regulating cardiomyocyte differentiation.^17,21-23 Furthermore, genome-wide association studies have revealed a genetic link between the human Casz1 locus and high blood pressure implicating CASZ1 in cardiovascular disease as well as development.²⁴ Through the use of Xenopus and human endothelial cell models, we have recently demonstrated that CASZ1 is also required for blood vessel development.¹⁷ In the absence of CASZ1, Xenopus embryos failed to generate a well-branched vascular network and lumen formation was impaired. At the cellular level, CASZ1-depletion by short hairpin RNA in human umbilical vein endothelial cells (HUVECs) resulted in the inability of cells to maintain adhesion to the underlying substrate and failure of cells to undergo the G1/S cell cycle transition; likely due to defective control of cell contractility. To elucidate the mechanism by which CASZ1 regulates these processes, we performed a cloning chromatin immunoprecipitation screen to identify direct transcriptional targets of CASZ1. One of the identified targets was EGFL7, a secreted ECM protein implicated in vessel morphogenesis.^25,26 We subsequently confirmed that CASZ1 is bound to Egfl7 and that Egfl7 was a bona fide CASZ1 target gene. Indeed, Egfl7 levels were downregulated in the absence of CASZ1, and depletion of EGFL7 in embryos and cells phenocopied the defects associated with loss of CASZ1 providing evidence that CASZ1 was required for transcriptional activation of Egfl7 for subsequent vessel development.

Although EGFL7 was previously implicated in vessel morphogenesis, the mechanism(s) by which EGFL7 regulated endothelial cell behaviors was unclear prior to these studies. Upon closer examination of CASZ1 and EGFL7-depleted HUVECs which displayed a thin, elongated morphology associated with impaired contractility of the trailing edge, we hypothesized that Rho GTPase signaling was dysregulated. In fact, staining with focal adhesion and F-actin markers revealed an absence of adhesions from sites of substrate contact and reduced, diffuse stress fibers reminiscent of RhoA inhibition.^27,28 Accordantly, levels of active RhoA and phosphorylation of non-muscle myosin II light chain were significantly diminished in CASZ1 and EGFL7-depleted cells. Somewhat surprisingly, total protein levels as well as transcript levels of RhoA were also greatly reduced under both conditions indicating that impairment of the RhoA pathway occurred at the level of gene transcription. Upon re-expression of Egfl7 in CASZ1-depleted cells, endothelial cells no longer detached from the substrate and proper localization of focal adhesions and stress fibers was restored indicating that RhoA lies downstream of CASZ1/Egfl7 (Fig. 1).

Figure 1. (A) The transcription factor CASZ1 directly binds to and activates expression of Egfl7 in endothelial cells. By a yet unknown mechanism, this transcriptional pathway promotes expression of the RhoA gene and subsequent GTPase activity which leads to proper adhesion of endothelial cells to the underlying substrate (as indicated by red lines). (B) In the absence of CASZ1 (or EGFL7), RhoA expression is diminished resulting in thin, elongated cells that cannot stably form adhesions resulting in their detachment from the underlying substrate.

As described above, regulation of RhoA activity by GEFs, GAPs, and RhoGDI is well-characterized and is presumed to be the main means by which RhoA activity is tightly controlled. However, it is well-documented that increased RhoA mRNA and protein expression are correlated with cancer progression, indicating that additional transcriptional layers of control likely exist and that it will be of importance to define the underlying mechanisms.^29-33 The only transcriptional pathway that has been shown to regulate the RhoA gene is the oncogenic Myc-Skp2-Miz-p300 complex which promotes RhoA expression in breast carcinoma cell lines.³⁴ Since Myc is required for proper embryonic development,³⁵ and abnormal development of blood vessels has been attributed to both overexpression and depletion of Myc,^36-38 it will be of future interest to examine whether the Myc/RhoA transcriptional network is also operative in endothelial cells and (if so) how this pathway might contribute to the EGFL7-dependent regulation of RhoA during vascular assembly and morphogenesis.

MicroRNAs have also been implicated in regulating RhoA transcript and protein abundance via direct binding to the 3′ UTR of the RhoA gene. Again, regulation of RhoA expression by these means has been highlighted in a number of cancer cell models wherein miRs 185, 31, and 155 have each been shown to impart tight control of RhoA mRNA and protein levels.^39-42 Importantly, miR-31 was also shown to target RhoA in cardiomyocytes, wherein it limited hypertrophic remodeling, indicating that regulation of RhoA by microRNAs is not restricted to oncogenesis and likely represents a common regulatory mechanism.⁴³ Therefore, it would be interesting to screen microRNA expression in CASZ1 or EGFL7-depleted cells as it is formally possible that aberrant microRNA activity downstream of CASZ1/Egfl7 may be responsible for the diminished RhoA mRNA and/or protein levels.

While the mechanism by which EGFL7 promotes RhoA transcription is currently unclear, recent studies have begun to shed light on how EGFL7 might initiate cell signaling. EGFL7 was shown to interact with lysyl oxidase (LOX) enzymes and to inhibit their ability to crosslink elastic fibers within the ECM, a process that imparts elasticity and resiliency to vessels.⁴⁴ Thus, it is possible that changes in the rigidity of the ECM alone could initiate mechanosensitive signals, or EGFL7-induced ECM remodeling could alter the accessibility/ binding affinity of cell-surface receptors responsible for mediating intracellular signaling cascades. Alternatively, EGFL7 may activate or antagonize signaling events by serving as a ligand for transmembrane receptors. For example, EGFL7 was recently shown to bind to and activate the integrin αvβ3 and this association was shown to be important for proper vessel morphogenesis and angiogenic sprouting.⁴⁵ While integrins have been intimately associated with mediating extracellular signals through activation of Rho GTPases,^46,47 less is understood about how modulation of integrins may affect their gene expression, though it remains a possibility that RhoA gene expression may be facilitated by positive feedback in response to increased RhoA activity.⁴⁸ EGFL7 has also been demonstrated to antagonize Notch signaling in endothelial cells as well as neural stem cells.^49,50 Again, however, it remains to be determined if RhoA is a direct or indirect downstream target of this pathway.

Although many questions still remain, our findings have uncovered a novel transcriptional pathway that may directly or indirectly promote RhoA gene expression. The depletion of CASZ1 or its direct target EGFL7 results in decreased RhoA mRNA, protein, and activity subsequently leading to improper endothelial cell behaviors associated with aberrant vascular development. These studies open up new models in which to probe the effects of dysregulated RhoA gene expression on cell behavior and to potentially provide insight into mechanisms directly controlling either RhoA gene expression or protein synthesis.

Charpentier MS, Christine KS, Amin NM, Dorr KM, Kushner EJ, Bautch VL, et al. CASZ1 promotes vascular assembly and morphogenesis through the direct regulation of an EGFL7/RhoA-mediated pathway. Dev Cell. 2013;25:132–43. doi: 10.1016/j.devcel.2013.03.003.