Ren, Bin

CD36 is a microvascular endothelial cell (MVEC) receptor mediating angiostatic activity of TSP-1 and related proteins. We previously reported that lysophosphatidic acid (LPA), a biologically active lipid signaling mediator, inhibited MVEC CD36 transcription via PKD-1 signaling. Moreover, CD36 transcriptional repression abolished endothelial cell responses to TSP-1 in vitro and in vivo . We now show with luciferase transfection assays that LPA exposure significantly suppressed CD36 promoter activity in MVEC. Co-exposure to class II HDAC inhibitors SAHA or TSA reversed the inhibition of promoter activity and restored transcription. HDAC7 knockdown by shRNA also attenuated LPA-suppressed CD36 promoter activity and mRNA levels. Mechanistically, FoxO1 was found to directly interact with putative FoxO1 binding sites (FHRE) in proximal promoter in vitro by avidin-biotin-conjugated DNA-binding assay, and in vivo by chromatin IP assay. Furthermore, FHRE mutations significantly attenuated the promoter activity. Western blots and immunofluorescence microscopy showed increased nuclear accumulation of PKD-1, FoxO1 and HADC7 after MVEC were exposed to LPA. Co-IP of nuclear extracts showed a physical interaction of HDAC7 with FoxO1 that was attenuated with PKD-1 knockdown. These data demonstrate that HDAC7 modulation by LPA receptor/PKD-1 signaling pathway represses FoxO1-mediated CD36 transcriptional activity. Additionally, angiogenic transcription profiling of endothelial cells revealed significantly increased mRNA expression of 17 angiogenic genes in response to LPA, including ephrin B2 expression that was confirmed by Western Blots. Transduction of constitutively active PKD-1 (PKD-CA) into EC also increased ephrin B2 expression. Functionally, PKD-CA overexpression promoted branching morphogenesis of arterial endothelial cells in a three-dimensional spheroid assay. The data indicate that PKD-1 is a potent angiogenic and arteriogenic kinase in EC by down-regulating CD36 transcription and promoting arteriogenesis. PKD-1-FoxO1-CD36 signaling axis may have potential as an important target in cardiovascular ischemia and malignant tumors.

Background: CD36 is a scavenger and antiangiogenic receptor that plays an important role in athero-thrombotic diseases, diabetes and cancer and contributes to obesity. Lysophosphatidic acid (LPA), a bioactive phospholipid signaling mediator, abolishes endothelial cell responses to antiangiogenic proteins containing thrombospondin type 1 homology domains by down-regulating endothelial CD36 transcription via protein kinase PKD-1 signaling. However, the precise mechanism as to how angiogenic signaling is integrated to regulate endothelial specific CD36 transcription remain unknown. Hypothesis: LPA represses CD36 transcription through PKD-1-mediated formation of a nuclear transcriptional complex in endothelial cells. Methods: Microvascular endothelial cells expressing CD36 were used for studying signaling and CD36 transcription by real time RT-qPCR, Western blotting, co-immunoprecipitation or avidin-biotin-conjugated DNA-binding assay; angiogenesis gene array was used for angiogenic gene profiling in response to LPA exposure. Spheroid-based angiogenesis assay, in vivo Matrigel assay and tumor angiogenesis model in CD36 deficiency and wild type mice were established to elucidate mechanisms of angiogenic signaling. Results: CD36 transcriptional repression involved PKD-1 signaling mediated formation of FoxO1-HDAC7 complex in the nucleus of endothelial cells. Unexpectedly, turning off CD36 transcription initiated reprogramming MVECs to express ephrin B2, a critical “molecular signature” involved in angiogenesis and arteriogenesis, and increased phosphorylation of Erk1/2, the MAP kinase important in arterial differentiation. PKD-1 signaling was also shown in tumor endothelium of Lewis lung carcinomas, along with low CD36 expression or CD36 deficiency. Angiogenic branching morphogenesis and in vivo angiogenesis were dependent on PKD-1 signaling. Conclusion: LPA/PKD1-HDAC7-FoxO1 signaling axis regulates endothelial CD36 transcription and mediates silencing of the antiangiogenic switch, resulting in proarteriogenic reprogramming. Targeting this signaling cascade could be a novel approach for cancer, diabetes, athero-thrombotic diseases and obesity.

Lysophosphatidic acid (LPA), a bioactive signaling phospholipid, down-regulates CD36 expression in microvascular endothelial cells (MVECs) via protein kinase PKD-1 signaling, thereby abolishing endothelial cell responses to its antiangiogenic ligand thrombospondin-1. However, little is known regarding mechanisms by which MVEC-specific CD36 transcription is regulated. We describe that in MVECs LPA represses CD36 transcription by activating a PKD-1 signaling that induces formation of a HDAC7/NCoR1/FoxO1 complex in the nucleus. Promoter analysis first identified FoxO1 as a transcription factor responsible for the CD36 transcription, which was confirmed by a chromatin-immunoprecipitation assay. Using a combination of PKD-1 gene transduction with co-immmunoprecipitation assay, we showed an increased interaction of HDAC7/NCoR1 with FoxO1 in response to LPA. However, HDAC7 and FoxO1 interaction was attenuated with PKD-1 silencing. Furthermore, based on results from an angiogenesis profiling with real time qPCR, doxycycline inducible constitutively active PKD-1 plasmids were transduced into tumor associated endothelial cells using a Lentiviral system to induce the PKD-1 expression. The results showed that turning off CD36 transcription reprograms by PKD-1 signaling was accompanied by an induced expression of ephrin B2 and activation of MAPK/ERK1/2 signaling, which are two critical “molecular signatures” involved in arteriogenesis. Moreover, three dimensional spheroid assay, a modified Boyden Chamber assay and in vivo Matrigel assay revealed that turning off CD36 transcription promoted angiogenesis in vitro and in vivo in a PKD-1-dependent manner. Immunofluorescence microscopy also showed the presence of this signaling pathway in the vasculature of Lewis lung carcinomas grown in cd36 deficient mice. In summary, our data suggest that a LPA-PKD-1-HDAC7/NCoR1-FoxO1 signaling axis is critical for transcriptional regulation of CD36 and mediates silencing of this antiangiogenic switch. This subsequently results in MVEC reprogramming for proangiogenic and arteriogenic responses. Therefore, targeting this signaling cascade could be a novel approach for malignant tumors, cardiovascular ischemia and other thrombotic diseases.

Objective— CD36 is a scavenger and antiangiogenic receptor that is important in atherothrombotic diseases, diabetes mellitus, cancer, and obesity. Lysophosphatidic acid, a phospholipid signaling mediator, abolishes endothelial cell responses to antiangiogenic proteins containing thrombospondin type 1 homology domains by downregulating endothelial CD36 transcription via protein kinase D1 (PKD-1) signaling. We aimed to understand mechanisms by which lysophosphatidic acid–mediated angiogenic signaling is integrated to regulate CD36 transcription and endothelial cell function via a nuclear transcriptional complex. Approach and Results— Microvascular endothelial cells expressing CD36 were used for studying angiogenic signaling and CD36 transcription. Gene transfection and transduction, RT-qPCR, avidin–biotin-conjugated DNA-binding assay, chromatin immunoprecipitation assay, co-immunoprecipitation, proximal ligation assay, and immunofluorescence microscopy showed that lysophosphatidic acid–mediated CD36 transcriptional repression involved PKD-1 signaling mediated formation of forkhead box protein O1–histone deacetylase 7 complex in the nucleus. Unexpectedly, turning off CD36 transcription initiated reprogramming microvascular endothelial cells to express ephrin B2, a critical molecular signature involved in angiogenesis and arteriogenesis. Spheroid-based angiogenesis and in vivo Matrigel angiogenesis assays indicated that angiogenic branching morphogenesis and in vivo angiogenesis were dependent on PKD-1 signaling. A mouse tumor angiogenesis model revealed enhanced PKD-1 signaling and expression of ephrin B2 and smooth muscle actin in neovessels of Lewis Lung Carcinomas, along with low-CD36 expression or CD36 deficiency. Conclusions— Lysophosphatidic acid/PKD-1 signaling leads to nuclear accumulation of histone deacetylase 7, where it interacts with forkhead box protein O1 to suppress endothelial CD36 transcription and mediates silencing of antiangiogenic switch, resulting in proangiogenic and proarteriogenic reprogramming. Targeting this signaling cascade could be a novel approach for ischemic cardiovascular disease and cancer.

Abstract CD36 is an endothelial cell (EC) receptor that mediates angiostatic activity and oxidative stress, which are important in ischemic disease and tumor progression. CD36 interacts with TSR domain proteins including thrombospondin-1. This results in a signal that is likely to initiate an antiangiogenic “switch," subsequently converting a growth factor-mediated proangiogenic response to an antiangiogenic response. However, in pathologic settings including malignant tumors, robust angiogenesis occurs despite the abundance of TSR-containing proteins in the microenvironment. This suggests that there is a mechanism by which TSR-mediated antiangiogenesis could be blunted via the localized downregulation of EC CD36 transcription. We previously reported that lysophosphatidic acid (LPA), a bioactive signaling phospholipid mediator, down-regulates microvascular EC (MVEC) CD36 transcription by protein kinase PKD-1 signaling via G protein coupled receptor LPA1,3. However, further elucidating the CD36 transcriptional mechanisms is critical to understanding the antiangiogenic switch. To this end, we recently discovered that obesity-derived LPA inhibited CD36 expression in tumor-associated ECs (TAECs). Similarly, inducing the constitutively active PKD-1 or PKD-CA expression also resulted in CD36 downregulation. Mechanistically, LPA treatment led to nuclear accumulation of PKD-1, HDAC7 and FoxO1. Furthermore, LPA-mediated PKD-1 signaling enhanced FoxO1-HDAC7 interaction in the nucleus. This interaction was attenuated with PKD-1 silencing. Intriguingly, doxycycline induced PKD-CA expression increased ephrin B2 expression and ERK activation, which are two critical “molecular signatures” involved in arteriogenesis. Functionally, FGF-2 induced a potent proangiogenic response even in the presence of TSP-1 in ECs pre-treated with LPA. Doxycycline induced PKD-CA expression stimulated robust arterial EC branching in a 3D spheroid assay. This angiogenic response was inhibited by a novel PKD-1 specific inhibitor. Moreover, in a mouse Lewis lung carcinoma model, we detected a minimal level of endothelial CD36 expression with robust tumor angiogenesis. The role of PKD-1 signaling in CD36 transcriptional repression and tumor arteriogenesis is a critical yet underexplored and underappreciated field. We propose that the LPA-PKD-FoxO1 signaling axis is essential for CD36 transcriptional repression and arteriogenic signaling and responses. The PKD-1 stimulated nuclear signaling network is critical to epigenetic suppression of CD36 transcription and silencing of CD36 antiangiogenic switch, subsequently turning on the angiogenic switch and stimulating the arteriogenic responses. Targeting the LPA-PKD1-FoxO1 signaling axis could have therapeutic potential in the ischemic disease and malignant tumors. Citation Format: Jacob D. Kohlenberg, Yiliang Chen, Brad Best, Peter Storz, Randall T. Peterson, Roy Silverstein, Bin Ren. A novel LPA-PKD1-FoxO1 pathway in endothelial cells provides an angiogenic switch via down-regulation of CD36 transcription and induction of arteriogenic responses . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-338. doi:10.1158/1538-7445.AM2013-LB-338