Endoglin: An 'Accessory' Receptor Regulating Blood Cell Development and Inflammation

Abstract

Transforming growth factor-β1 (TGF-β1) is a pleiotropic factor sensed by most cells. It regulates a broad spectrum of cellular responses including hematopoiesis. In order to process TGF-β1-responses in time and space in an appropriate manner, there is a tight regulation of its signaling at diverse steps. The downstream signaling is mediated by type I and type II receptors and modulated by the 'accessory' receptor Endoglin also termed cluster of differentiation 105 (CD105). Endoglin was initially identified on pre-B leukemia cells but has received most attention due to its high expression on activated endothelial cells. In turn, Endoglin has been figured out as the causative factor for diseases associated with vascular dysfunction like hereditary hemorrhagic telangiectasia-1 (HHT-1), pre-eclampsia, and intrauterine growth restriction (IUPR). Because HHT patients often show signs of inflammation at vascular lesions, and loss of Endoglin in the myeloid lineage leads to spontaneous inflammation, it is speculated that Endoglin impacts inflammatory processes. In line, Endoglin is expressed on progenitor/precursor cells during hematopoiesis as well as on mature, differentiated cells of the innate and adaptive immune system. However, so far only pro-monocytes and macrophages have been in the focus of research, although Endoglin has been identified in many other immune system cell subsets. These findings imply a functional role of Endoglin in the maturation and function of immune cells. Aside the functional relevance of Endoglin in endothelial cells, CD105 is differentially expressed during hematopoiesis, arguing for a role of this receptor in the development of individual cell lineages. In addition, Endoglin expression is present on mature immune cells of the innate (i.e., macrophages and mast cells) and the adaptive (i.e., T-cells) immune system, further suggesting Endoglin as a factor that shapes immune responses. In this review, we summarize current knowledge on Endoglin expression and function in hematopoietic precursors and mature hematopoietic cells of different lineages.

Keywords: CD105; Endoglin; TGF-β-signaling; adaptive immunity; hematopoiesis; hematopoietic stem cells; innate immunity.

Figure 1

Endoglin biology in endothelial cells. (central part) Endoglin (Eng) is a component of a receptor complex in endothelial cells comprising the type I receptor dimers activin receptor-like kinase 5 (ALK5) or ALK1, and type II receptor dimers binding ligands, e.g., transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein 9 (BMP9), of the TGF-β-superfamily. Interaction with these receptors either activates Smad1/5/8 or Smad2/3 signaling (phosphorylation) to regulate corresponding target genes, e.g., inhibitor of differentiation 1 (Id1) and plasminogen activator inhibitor-1 (PAI-1), and finally, cellular responses including proliferation and migration to balance the activation and resolution phase of angiogenesis. Localization of Eng in caveolin-1 (cav-1) positive lipid rafts mediates interaction of heat shock protein 90 (Hsp90) with endothelial NO-synthase (eNOS) regulating its function. (upper part) Beside the long variant of Eng (L-Eng), a C-terminally truncated, shorter splice variant (S-Eng) and a shedded form, mediated by matrix metalloproteinase (MMP)-14, comprising only the extracellular domain (sEng) has been identified. All three variants of Eng are involved individually in cellular (senescence) and pathological conditions (e.g., HHT-1, pre-eclampsia), respectively. The extracellular domain of Eng is N- and O-glycosylated and human Eng contains an integrin binding motif (RGD) motif. The L-Eng C-terminal domain is a substrate for TGF-β receptors leading to serine/threonine phosphorylation. Tyrosine phosphorylation is mediated by Src. L-Eng interacts with its cytoplasmic domain with several intracellular proteins regulating receptor trafficking, phosphorylation/activity of kinases, and focal adhesion amongst others. The functional difference of S-Eng compared to L-Eng most likely arises from the missing binding- and phosphorylation-sites in its C-terminus. Abbreviations used are: GIPC, GAIP C-terminus-interacting protein; HHT-1, hereditary hemorrhagic telangiectasia 1; Smad, small mothers against decapentaplegic; pERK1/2, phosphorylated extracellular signal-regulated kinase 1/2; PI3K/Akt, phosphatidylinositol 3-kinase; TβRI/II, transforming growth factor-β1 type I or II receptor; ZRP-1, zyxin-related protein-1.

Figure 2

Endoglin expression and function in fetal hematopoietic cells. (A) Endoglin (Eng) expression is detected as early as embryonic day 7.5 (E7.5) in mesodermal cells (Flk-1⁺) and primitive erythroblasts (EryP, GATA1⁺) of the blood islands in wild-type mice. Endoglin expressing cells comprise all hematogenic and endothelial potential. In line, Eng deficiency (Eng^−/−) resulted in reduced EryP colonies and at E9.5 reduced erythroblasts (CD71⁺/Ter119⁺) in the yolk sac (YS) and diminished numbers of BFU-E and GEMM progenitors. (B) (upper) Initially it was shown in the development of embryoid bodies, by culturing ES cells on OP9 feeder cells, that Endoglin is transiently expressed during the progression from Flk-1⁺/CD45⁺ to the Flk-1⁻/CD45⁺ stage. Using Endoglin deficient ES cells it gets evident that although Flk-1 mesodermal precursor differentiation is normal, the CD45⁺ subset of hematopoietic cells is reduced at d9. In addition, the differentiation of ES cells into erythroid (CD45⁻/Ter119⁺) and myeloid (CD45⁺/CD11b⁺) was severely diminished along with a strongly reduced adult β-globin gene expression indicating impaired definitive erythropoiesis. (lower) Another study showed CD105 expression already in ES cells and Eng deficiency affected the blast-colony forming cell (BL-CFC) frequency and lowered its hematogenic potential, while increasing endothelial potential. EryP frequency was reduced in line with reduced expression of GATA1, embryonic and adult globin. The afore mentioned results were confirmed by using ES cells with doxycycline-inducible overexpression of Eng which leads to revers effects in comparison to the above described hematopoietic defects.

Figure 3

Endoglin expression and function in adult hematopoietic cells. (A) Bone marrow: In the adult KLS⁺ (Kit⁺/Lin⁻/Sca-1⁺) compartment, Eng is highest expressed in long term repopulating hematopoietic stem cells (LT-HSC). Expression in short term (ST-HSC) and multipotent progenitors (MPP) is much lower. (B) In the adult KLS⁻ (Kit⁺/Lin⁻/Sca-1⁻) compartment, Eng expression is low in the common myeloid (CMP) and lymphoid progenitors (CLP). Of the committed precursor cells, CD105 is low expressed in the granulocyte-monocyte progenitor (GMP) but highly expressed in the megakaryocyte-erythroid progenitor (MEP). Using a different FACS panel including CD105, it could be shown that Endoglin is transiently high expressed in pre-CFU-E and CFU-E. (C) peripheral blood: In adult circulating cells, CD105 could not be phenotypically detected in erythrocytes, thrombocytes and granulocytes. However, weak expression was shown in CD14⁺ monocytes and subsets of T- and B-cells. (D) Of the tissue residing cells, CD105 is expressed in M1 and M2 polarized macrophages as well as in tryptase positive mast cells. (E) A subset of circulating CD34⁺ co-expresses CD105. Those progenitor cells contain primitive precursors with a high frequency of long-term culture initiating cells (LTC-IC). In addition, these cells show no colony formation, except megakaryocyte colonies, unless TGF-β1 is blocked. (F) By treatment of bone marrow cells with 5-fluorouracil (block of cell cycle), HSC are enriched. Manipulation of Eng expression in those myeloid cells, i.e., shRNA knock-down or lentiviral overexpression, showed that Eng blocks TGF-β1-mediated inhibition of proliferation. Moreover, analysis of recipient bone marrow indicated that Eng has no impact on white blood lineages but has a negative impact on erythroid burst-forming unit (BFU-E) formation and erythroid differentiation (reduction of basophilic erythroblasts, CD71⁺/Ter119⁺).

Figure 4

Transcript analysis of endoglin expression in peripheral blood cells. Transcript analysis of peripheral blood cells verified the very low presence of endoglin in granulocytes, low expression in T- and B-cells and showed a high expression in monocytes. In addition, dendritic cells express considerable amounts of Eng mRNA. Expression data were taken from the entry for Endoglin in the Human Protein Atlas [101].

Figure 5

Endoglin expression and function in macrophages. (A) Bone marrow-derived pro-monocytes differentiate to monocytes which enter the circulation. (A,B) Monocytes extravasate, enter tissues, differentiate to macrophages, and are polarized to anti- (M2) or pro-(M1) inflammatory macrophages upon the appropriate stimuli, of which both express Endoglin (Eng). In the course of these processes L-Eng is upregulated. Monocyte-derived dendritic cells have not been analyzed for Eng expression so far. Cellular aging and oxidative stress causes senescence accompanied by proportional higher upregulation of S-Eng. (C) M1 polarized (pro-inflammatory) macrophages are pro-inflammatory and anti-angiogenic. This is in part due to the secretion of MMP-12 which is missing in the secretome of M2 macrophages. Secretion of MMP-12 causes shedding of Eng in the membranes of macrophages and EC leading to the liberation of sEng, which is an anti-angiogenic molecule. (D) For functional analysis, U937 L- and S-Eng transfectants were analyzed by proteome [135] and transcriptome [133] analysis. In line, both studies showed a reduced expression of different integrin subunits in both transfectants accompanied by reduced adhesion. However, the data of Aristorena and colleagues [135] suggested that S-Eng blocks M1 and favors M2 polarization, whereas the L-Eng data were not that clear. Both studies showed in accordance that L-Eng up- and S-Eng down-regulates the inhibinBα (activin A) subunit. (E) A myeloid lineage-specific Eng knockout showed spontaneous infections and a lower inflammatory response in vivo coupled to compromised inflammatory macrophage functions in vitro, implying less M1 activity in the absence of Eng. Abbreviations used: CCR2, C-C chemokine receptor type 2; DC, dendritic cell; EC, endothelial cell; Eng, Endoglin; GM-CSF, granulocyte-macrophage colony-stimulating factor; IL, interleukin; LPS, lipopolysaccharide; M-CSF, macrophage colony-stimulating factor; MMP, matrix metalloproteinase; NOS2, inducible NO-synthase; PMA, Phorbol-12-myristate-13-acetate.

Figure 6

Endoglin expression and function in T cells. (A) Adipose tissue derived stem cells (ASC) are able to regulate T-cell dependent immune responses in part by sequestering reactive CD4⁺ T-cells. Allo-activated CD4⁺ T cells tightly bound to ASC show a higher CD25 expression and in turn IL-2-dependent STAT5 activation. These cells present a higher proliferation and increased Endoglin (Eng) expression along with a higher Smad1/5/8 phosphorylation (activation). Nevertheless, aside the higher Foxp3 expression, the bound cells also had a higher CD127 expression excluding that they are Tregs. (B) High expression of Eng is present in CD4⁺ T-cells isolated from peripheral blood. TCR/pathway activation of these CD4⁺ T-cells by CD3 or PMA leads to a higher surface expression of Eng and increased serine/threonine phosphorylation of the receptor. Activation of Eng induces ERK1/2 phosphorylation and higher proliferation. On the other hand, Eng interferes with TGF-β1-mediated Smad signaling and inhibition of proliferation. (C) In the setting of tumorigenesis, intratumoral CD4⁺ T-cells, i.e., CD25⁺/Foxp3⁺ Tregs, express higher Eng compared to their circulating counterparts. This causes an antibody-dependent cellular cytotoxicity (ADCC) resulting in reduction of intratumoral Tregs (immunosuppressive) upon application of TRC105 via CD8⁺ T-cells.