Vascular cell responses to exogenous heparin have been documented to include decreased vascular smooth muscle cell proliferation following decreased ERK pathway signaling. However, the molecular mechanism(s) by which heparin interacts with cells to induce those responses has remained unclear. Previously characterized monoclonal antibodies that block heparin binding to vascular cells have been found to mimic heparin effects. In this study, those antibodies were employed to isolate a heparin binding protein. MALDI mass spectrometry data provide evidence that the protein isolated is transmembrane protein 184A (TMEM184A). Commercial antibodies against three separate regions of the TMEM184A human protein were used to identify the TMEM184A protein in vascular smooth muscle cells and endothelial cells. A GFP-TMEM184A construct was employed to determine colocalization with heparin after endocytosis. Knockdown of TMEM184A eliminated the physiological responses to heparin, including effects on ERK pathway activity and BrdU incorporation. Isolated GFP-TMEM184A binds heparin, and overexpression results in additional heparin uptake. Together, these data support the identification of TMEM184A as a heparin receptor in vascular cells.For more than 30 years, heparin has been known to specifically bind to cells in the vasculature and alter their physiology in addition to its well recognized function as an anticoagulant. Heparin binds to many proteins, including numerous growth factors, cytokines, coagulation factors, cell adhesion molecules, growth factor receptors, matrix glycoproteins, and others (for a review, see Ref. 1). In fact, heparin and the closely related glycosaminoglycan heparan sulfate (HS), 4 interact with more than 400 proteins (2). Heparin decreases endothelial cell (EC) inflammatory gene expression and slows vascular smooth muscle cell (VSMC) proliferation (reviewed in Ref. 3). Specifically, ECs bind and endocytose heparin (4, 5), which is followed by decreased inflammatory signaling through NF-B (6) and stress kinase activity (7,8). Heparin binding in VSMCs (9) results in decreases in growth factor-induced ERK signaling (10, 11), inhibition of downstream transcription factor activity (12-14), changes in cell cycle inhibitory factors (15), and decreased proliferation (10, 16).Reports of fluorescent heparin uptake into cells, where it modulated transcription factor function (17), and the requirements of HSPGs for basic growth factor delivery to the nucleus (18) indicate that receptor-mediated uptake of heparin or HS may also be critical for some heparin effects. Similarly, shed HSPG syndecan-1 can be taken up by cells and transported to the nucleus, where it alters histone acetylation (19). HS chains are required for uptake, and this uptake can be inhibited by exogenously added heparin. It is likely that the uptake of highly charged heparin and HS chains involves a receptor to manage transport across the membrane. Although many heparin-interacting proteins have been linked to specific functions, a receptor respon...
Despite the large number of heparin and heparan sulfate binding proteins, the molecular mechanism(s) by which heparin alters vascular cell physiology is not well understood. Studies with vascular smooth muscle cells (VSMCs) indicate a role for induction of dual specificity phosphatase 1 (DUSP1) that decreases ERK activity and results in decreased cell proliferation, which depends on specific heparin binding. The hypothesis that unfractionated heparin functions to decrease inflammatory signal transduction in endothelial cells (ECs) through heparininduced expression of DUSP1 was tested. In addition, the expectation that the heparin response includes a decrease in cytokine-induced cytoskeletal changes was examined. Heparin pretreatment of ECs resulted in decreased TNF␣-induced JNK and p38 activity and downstream target phosphorylation, as identified through Western blotting and immunofluorescence microscopy. Through knockdown strategies, the importance of heparin-induced DUSP1 expression in these effects was confirmed. Quantitative fluorescence microscopy indicated that heparin treatment of ECs reduced TNF␣-induced increases in stress fibers. Monoclonal antibodies that mimic heparin-induced changes in VSMCs were employed to support the hypothesis that heparin was functioning through interactions with a receptor. Knockdown of transmembrane protein 184A (TMEM184A) confirmed its involvement in heparin-induced signaling as seen in VSMCs. Therefore, TMEM184A functions as a heparin receptor and mediates anti-inflammatory responses of ECs involving decreased JNK and p38 activity.For almost 100 years heparin has been used as an anticoagulant. Specific heparin interactions with proteins important in the anti-clotting system are now well understood. Many heparin binding proteins, including quite a few involved in modulating vascular function, inflammation, and angiogenesis, have been identified (reviewed in Ref. 1). The large number of reports indicating evidence of decreased endogenous heparin and heparan sulfates (HS) 3 in atherosclerosis (in model animals and human disease) led to a proposal that decreases in endogenous heparins might be important in the development of atherosclerosis (2). More recent evidence in support of that hypothesis includes increased heparanase expression in atherosclerosis (reviewed in Ref.3) and increased levels of glycocalyx heparan sulfate in regions of the vasculature where laminar flow decreases the likelihood of atherosclerosis development (4).In addition to heparin, heparin binding proteins typically also bind HS chains on HS proteoglycans (HSPGs). Although the carbohydrate backbones of heparin and HS are identical, modifications and sulfation patterns vary. HS chains have fewer sulfate residues per disaccharide and a lower overall charge, but their widespread expression suggests that HS may provide many in vivo functions identified originally as heparin functions (reviewed in Ref. 5). Heparin binding to growth factors modulates their activity and appears to protect them from degradation...
Transmembrane protein 184A (TMEM184A) was recently identified as the heparin receptor in vascular cells. Heparin binds specifically to TMEM184A and induces anti-proliferative signaling in vitro. Though it is highly conserved, the physiological function of TMEM184A remains unknown. The objective of this study was to investigate the expression and effects on vascular regeneration of TMEM184A using the adult zebrafish regenerating caudal fin as an in vivo model. Here, we show that Tmem184a is expressed in vascular endothelial cells (ECs) of mature and regenerating zebrafish fins. Transient morpholino (MO)-mediated knockdown of Tmem184a using two validated MOs results in tangled regenerating vessels that do not grow outward and limit normal overall fin regeneration. A significant increase in EC proliferation is observed. Consistent with in vitro work with tissue culture vascular cells, heparin has the opposite effect and decreases EC proliferation which also hinders overall fin regeneration. Collectively, our study suggests that Tmem184a is a novel regulator of angiogenesis.
When novel proteins are identified through affinity-based isolation and bioinformatics analysis, they are often largely uncharacterized. Antibodies against specific peptides within the predicted sequence allow some localization experiments. However, other possible interactions with the antibodies often cannot be excluded. This situation provided an opportunity to develop a set of assays dependent on the protein sequence. Specifically, a construct containing the gene sequence coupled to the GFP coding sequence at the C-terminal end of the protein was obtained and employed for these purposes. Experiments to characterize localization, ligand affinity, and gain of function were originally designed and carried out to confirm the identification of TMEM184A as a heparin receptor. In addition, the construct can be employed for studies addressing membrane topology questions and detailed protein-ligand interactions. The present report presents a range of experimental protocols based on the GFP-TMEM184A construct expressed in vascular cells that could easily be adapted for other novel proteins.
Endothelial cells (ECs) comprise the innermost layer of blood vessels and serve numerous critical roles in the vasculature, including nutrient delivery, gas exchange, leukocyte trafficking, and immunological surveillance. Additionally, ECs of blood vessels are constantly exposed to fluid shear stress (FSS) in the form of blood flow, and maintain vessel tone by sensing and responding to FSS through a process called mechanotransduction. Mechanotransduction in ECs is a complex biological process that involves several key adherens junction proteins, such as PECAM‐1, VE‐cadherin, and the vascular endothelial growth factor (VEGF) receptor, VEGFR2. There also exist many other proteins that act as co‐factors to regulate mechanotransduction, such as integrins and heparan sulfate proteoglycans (HSPGs). ECs typically respond to the mechanical forces of laminar (normal) FSS by aligning in the direction of flow, elongating, and maintaining communication at adherens junctions between cells. These responses constitute endogenous atheroprotective mechanisms that ECs use to prevent abnormal vascular physiology. Additionally, regions of the vasculature exposed to laminar blood flow have higher levels of the vasodilator nitric oxide (NO), an important vascular signaling molecule that is produced by endothelial nitric oxide synthase (eNOS). Improper mechanosensing, combined with chronic inflammation or disturbed FSS, can lead to EC dysfunction, which accumulates over time and results in the progression of vascular diseases such as atherosclerosis. Our laboratory recently identified Transmembrane Protein 184A (TMEM184A) as a heparin receptor in vascular cells. Moreover, we have since found that TMEM184A is a modulator of vascular regeneration in vivo. Here, we present evidence that TMEM184A is also involved in EC responses to FSS that prevent dysfunction. Immunofluorescence and immunoprecipitation data collectively suggest that TMEM184A colocalizes and interacts with the classical mechanosensors, several of the HSPGs, and eNOS on the surface of static ECs. Under laminar FSS, TMEM184A colocalization with VE‐cadherin and VEGFR2 is even more apparent. Degradation of endogenous heparan sulfates with heparinase drastically changes typical EC responses to laminar FSS by altering the normal pattern of TMEM184A accumulation in response to shear and impairing EC elongation and alignment. Both of these responses are partially recovered by the addition of heparin. ECs that overexpress TMEM184A have enhanced VE‐cadherin expression at adherens junctions after acclimation to laminar FSS. Treatment of ECs with VEGF to stimulate VEGFR2 produces substantial changes in TMEM184A dynamics and internalization under FSS conditions. Together, these data support a role for TMEM184A in mediating EC responses to laminar flow. Further research is necessary to confirm this role for the heparin receptor in order to assess potential applications for the treatment of cardiovascular disease.Support or Funding InformationNIH HL54269This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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