PULMONARY ARTERIAL HYPERTENSION AND HIV-INFECTION: pulmonary hypertension and hiv infection is there an association?
Pulmonary Hypertension (PH) is an increase of blood pressure in the pulmonary artery, pulmonary vein, or pulmonary capillaries, together known as the lung vasculature, leading to shortness of breath, dizziness, fainting, and other symptoms, all of which are exacerbated by exertion. Pulmonary hypertension can be a severe disease with a markedly decreased exercise tolerance and heart failure.
HIV infection is considered a risk factor for the development of pulmonary arterial hypertension (PAH).
Intersection and interactions between a genetic predisposition involving the BMPR2 signaling pathway and an impaired metabolic and chronic inflammatory state in the vessel wall.
Members of the TGF-beta superfamily, including TGFB, BMPs, and activin, transduce signals by binding to heteromeric complexes of type I and II serine/threonine kinase receptors, leading to transcriptional regulation by phosphorylated Smads. The BMPR2 and ACVRL1 genes encode type II and type I serine/threonine kinase receptors, respectively. Mutation in the SMAD9 gene (also known as SMAD8) suggests that downregulation of the downstream TGFB/BMP signaling pathway may play a role in primary pulmonary hypertension (International PPH Consortium et al., 2000; Shintani et al., 2009).
The International PPH Consortium et al. (2000) and Deng et al. (2000) showed that PPH1 is caused by mutations in the BMPR2 gene. These BMPR2 mutations were found in 7 of 8 families exhibiting linkage to markers adjacent to BMPR2 by the International PPH Consortium et al. (2000) and in 9 of 19 of the families exhibiting linkage and/or haplotype sharing with markers adjacent to BMPR2 by Deng et al. (2000). Both groups found that the BMPR2 mutations are heterogeneous and include termination, frameshift, and nonconservative missense changes in amino acid sequence. By comparison with in vitro studies, the International PPH Consortium et al. (2000) predicted that the identified BMPR2 mutations would disrupt ligand binding, kinase activity, and heteromeric dimer formation.
Eddahibi et al. (2001) reported that pulmonary artery smooth muscle cells (SMCs) from patients with PPH grew faster than those from controls when stimulated with serum or serotonin, due to increased expression of 5-HTT. Inhibitors of 5-HTT attenuated the growth-stimulatory effects of serum and serotonin. Expression of 5-HTT was increased in cultured pulmonary artery SMCs as well as in platelets and lungs from patients with PPH, where it predominated in the media of thickened pulmonary arteries and in onion bulb lesions. The L allele variant of the 5-HTT promoter, which is associated with 5-HTT overexpression and increased pulmonary artery SMC growth, was present in homozygous form in 65% of PPH patients but in only 27% of controls (p less than 0.001). Eddahibi et al. (2001) concluded that 5-HTT activity plays a key role in the pathogenesis of pulmonary artery SMC proliferation in PPH and that a 5-HTT polymorphism confers susceptibility to PPH.
Thomson et al. (2000) analyzed the BMPR2 gene in 50 unrelated patients with sporadic PPH and identified 11 different heterozygous mutations in 13 of the 50 PPH patients, including 3 missense, 3 nonsense, and 5 frameshift mutations. Analysis of parental DNA was possible in 5 cases and showed 3 occurrences of paternal transmission and 2 of de novo mutation of the BMPR2 gene. Thomson et al. (2000) noted that because of low penetrance, in the absence of detailed genealogic data, familial cases may be overlooked.
Humbert et al. (2002) analyzed the BMPR2 gene in 33 unrelated patients with sporadic PPH and 2 sisters with PPH, all of whom had taken fenfluramine derivatives. Three BMPR2 mutations were identified in 3 (9%) of the 33 unrelated patients, and a fourth mutation was identified in the 2 sisters. Mutation-positive patients had similar clinical and hemodynamic characteristics when compared to mutation-negative patients, except for a shorter duration of exposure to fenfluramine derivatives before illness (median exposure, 1 month and 4 months, respectively). Humbert et al. (2002) concluded that BMPR2 mutations may combine with exposure to fenfluramine derivatives to greatly increase the risk of developing severe pulmonary arterial hypertension.
NORMAL BMP/TGF-β SIGNALING
and were originally identified as molecules regulating growth and differentiation of bone and cartilage. BMPs regulate growth, differentiation, and apoptosis in a diverse number of cell lines, including mesenchymal and epithelial cells, acting as instructive signals during embryogenesis and contributing to the maintenance and repair of adult tissues. . Although the short form of the receptor is widely expressed in human tissues, it is not known whether this form of the receptor serves a differential function compared with the long form. BMPR-II initiates intracellular signaling in response to specific ligands: BMP-2, BMP-4, BMP-6, BMP-7, growth and differentiation factor-5 (GDF-5), and GDF-6. Ligand specificity for different components of the receptor complex are emerging that may have functional significance to the tissue-specific nature of BMP signaling. The extreme tissue specificity of BMP signaling is highlighted by the diverse human diseases associated with mutations in type I and II receptors. Although BMPR-II mutation is associated with FPAH, BMPRIA mutation causes familial juvenile colonic polyposis, and BMPRIB mutation causes hereditary brachydactyly. The majority of ligands (BMP-2, BMP-4, BMP-7, and GDF-5 and GDF-6) bind with high affinity to the type I receptors, predominantly BMPRIA activin-like receptor kinase-3 (ALK-3) or BMPRIB (ALK-6), and with very low affinity to BMPR-II. GDF-5 demonstrates specificity for BMPRIB. In contrast, BMP6 binds with high affinity to BMPR-II. After ligand binding, the type II receptor phosphorylates a glycine-serine rich domain on the proximal intracellular portion of an associated type I receptor. Conformational changes that occur in the ligand-receptor complex when both receptor types contacting the ligand are required for cross-linking of the ligand to BMPR-II and intracellular signal transduction.
. BMPs signal via a restricted set of receptor-mediated Smads (R-Smads), Smads 1, 5, and 8, which must complex with the common partner Smad (co-Smad), Smad-4, to translocate to the nucleus. TGF-β and activins signal via a different set of R-Smads, Smads 2 and 3. Target gene transcription is regulated by a variety of mechanisms, including direct binding of the Smad complex to DNA, interaction with other DNA proteins (e.g., activator protein-1 [AP-1] and transcription factor E-3), and recruitment of transcriptional coactivators or co-repressors . Switching off Smad signaling in the cell is achieved via Smad ubiquitination and regulatory factors (Smurfs) and by recently identified Smad phosphatases.
THE CONSEQUENCES OF BMPR2 MUTATION FOR BMP/TGF-β SIGNALING
Two recent studies have shown that the mechanism by which BMPR-II mutants disrupt BMP/Smad signaling is heterogeneous and mutation specific. Of the missense mutations, substitution of cysteine residues within the ligand binding or kinase domain of BMPR-II leads to reduced trafficking of the mutant protein to the cell surface, a process that may also interfere with BMP type I receptor trafficking. In contrast, noncysteine mutations within the kinase domain reach the cell surface but fail to activate Smad-responsive luciferase reporter genes due to an inability to phosphorylate BMP type I receptors. Mutations in the ligand binding and kinase domains exhibit a dominant-negative effect on wild-type receptor function in terms of Smad signaling. Interestingly, BMPR-II mutants with missense mutations involving the cytoplasmic tail are able to traffic to the cell surface and are capable of activating Smad-responsive luciferase reporter genes to some extent but are almost certainly relatively deficient in their ability to transduce signals via Smads. In addition, pulmonary artery smooth muscle cells from mice heterozygous for a null mutation in the BMPR2 gene are deficient in Smad signaling. Thus, haploinsufficiency or missense mutation seems to lead to a loss of signaling via the Smad1/5 pathway. One study has reported that marked siRNA knockdown of BMPR-II leads to increased Smad signaling in response to some ligands (e.g., BMP7). The significance of this observation remains to be determined. Another potential gain of function as a consequence of BMPR-II mutation was reported in a mouse epithelial cell line. In these cells, transfection with BMPR-II mutant constructs led to ligand-independent activation of p38MAPK. Furthermore, these cells showed enhanced serum-induced proliferation as compared with wild-type transfected cells; this abnormal proliferation was abolished by treatment with a selective p38MAPK inhibitor. Subsequently, however, we were unable to find evidence for constitutive activation of MAPK pathways in pulmonary artery smooth muscle cells (PASMCs) isolated from patients with FPAH or in cells from heterozygous BMPR-II knockout mice. The constitutive activation of MAPK pathways by mutant BMPR-II may be cell-type specific. Further studies are needed to elucidate how BMPR-II mutation affects on MAPK signaling.
Our group has previously shown that PASMCs isolated from patients with IPAH or FPAH exhibit an exaggerated growth response to TGF-β1. TGF-β1 is not a ligand for the BMP receptors. In addition, the abnormal response to TGF-β does not seem to be caused by alterations in the expression of TGF-β type I, II, or III receptors. Mutations in the type I TGF-β receptor, ALK-1, have been observed in patients with severe PAH occurring in families with hereditary hemorrhagic telangiectasia. ALK-1 is unusual among the TGF-β receptors in that it signals via Smad1/5 rather than Smad2/3. This highlights the potential importance of a loss of Smad1/5 signaling in the vasculature as a cause of pulmonary vascular remodeling. However, a further important functional consequence of loss of Smad1/5 signaling may be a gain of TGF-β signaling via Smad2/3. Support for this concept comes from experiments in endothelial cells designed to elucidate the diverse responses of cells to TGF-β. In endothelial cells, Smad1/5 functionally antagonizes Smad2/3 signaling. This may be because Smad1/5 signaling competes for availability of the co-Smad, Smad4. In addition, Smad1 may physically interact with Smad3 and lead to degradation or prevent phosphorylation. This antagonism between TGF-β and BMP signaling pathways provides a mechanism for their often-observed functional antagonism in diverse settings. For example, BMP7 can antagonize the epithelial–mesenchymal cell transition induced by TGF-β. BMP7 inhibits TGF-β–dependent renal fibrosis in animal models. In cultured cells BMPs can antagonize TGF-β–induced COX-2 expression and TGF-β–dependent myofibroblast transformation. Thus, failure of BMP signaling via Smad1/5 can increase TGF-β/ALK-5/Smad2/3 signaling, which may be part of the molecular switch that determines the altered responsiveness to TGF-β. In fibroblasts, the profibrotic response to TGF-β is partly due to activation of the Abelson kinase c-Abl, a target of the drug imatinib, which has been recently shown to be effective in a case of FPAH. In addition, TGF-β is known to increase expression of the platelet-derived growth factor (PDGF) receptor, particularly in the context of scleroderma. Imatinib is also an effective PDGF receptor kinase inhibitor. The potential importance of this is that strategies aimed at reducing TGF-β/PDGF signaling become rational and realistic therapeutic goals in the treatment of FPAH
he constitutively active kinase domains of type II receptors phosphorylate type I receptors, and this in turn activates the SMAD signaling pathway through phosphorylation of receptor SMADs (SMAD1, SMAD5 and SMAD8). These associate with co-SMADs (SMAD4) to form a heteromeric complex that translocates to the nucleus and stimulates the expression of a wide range of target genes, including the gene encoding the iron regulatory peptide hepcidin. BMPs can also signal through SMAD-independent pathways, notably via MAP kinases. Dorsomorphin inhibits BMP signaling through the SMAD pathway, likely by affecting BMPR-I kinase activity. Many of the previously known inhibitors of BMP signaling (such as noggin and chordin) act upstream to sequester BMPs and cannot differentiate SMAD-dependent from SMAD-independent signaling. The activation of the hepcidin gene by IL-6 requires both the JAK-STAT and BMP-SMAD pathways, but how the pathways interact is unclear. Similarly, TfR2 and the HFE–TfR1 complex can alter hepcidin expression, but it is not known whether their functions require the BMP-SMAD system.
Figure "BMP signaling"
HIV Tat-specific factor 1 is a protein that in humans is encoded by the HTATSF1 gene.
Whereas most DNA sequence-specific transcription factors increase the rate of initiation and interact with enhancer or promoter DNA, human immunodeficiency virus-1 (HIV-1) Tat predominantly stimulates elongation and interacts with the trans-acting responsive (TAR) RNA element. Tat is essential for HIV replication.[supplied by OMIM]
Functionally, Tat is an essential, potent stimulator of HIV-1 transcription from the viral promoter as well as suppressor of the immune response through interaction with host transcription factors. HIV-1 transcription is up-regulated significantly by the interaction of Tat with host nuclear proteins such as cyclin T1, facilitating enhanced elongation of the viral transcript. Structurally, Tat possesses the canonical transactivator characteristics including a nucleic acid-binding domain and activation domain. Full-length Tat protein is encoded by two exons, resulting in an 86- or 101-amino acid product, depending on the viral isolate. Later, when HIV-1 infection becomes less efficient, synthesis of the truncated, one-exon product is observed. However, both Tat proteins up-regulate HIV-1 transcription.
Regulation of HIV-1 Transcription – Once the HIV genome is integrated into the host genome it is transcribed by RNA polymerase II and expression of viral genes is controlled by P- TEFb. HIV takes control of the P-TEFb control process through the use of a small viral protein, Tat, that takes P- TEFb from the 7SK snRNP and delivers it to the TAR element in the 5' end of the nascent HIV transcript. In collaboration with the Tahirov lab (UNMC) Department of Biochemistry (University of Iowa), recently solved the structure of an HIV Tat - P-TEFb complex. This structure should be useful in developing anti-HIV treatments that do not target the uncomplexed P-TEFb.
HIV Tat represses transcription of BMPR2
Previous studies have shown that Tat represses transcription of the mannose receptor in macrophages, as well as major histocompatibility complex (MHC) classes I and II in T lymphocytes. To determine if Tat represses BMPR2 transcription in macrophages, a 1725-bp BMPR2 (–1725) promoter driving the luciferase reporter gene was transiently cotransfected with Tat expression vector or empty vector into the human U937 monocytic cell line. . Transcription from the CMV promoter was unaffected by Tat, and Tat significantly enhanced transcription from the HIV promoter or LTR. An approximate tenfold induction from the LTR was observed when the amount of Tat expression vector was increased (data not shown). Tat also repressed transcription from the mannose receptor promoter as described previously. When the increasing Tat expression vector was cotransfected with the BMPR2 promoter reporter construct, a dose-dependent repression of BMPR2 promoter activity was determined, with a maximum inhibition of 80% with 2 μg Tat vector. Tat protein expression in these transfection experiments was verified by Western blotting as described previously.
HIV-1 Tat-mediated repression from the BMPR2 promoter is localized to the first 208 bp
To localize the region in the BMPR2 promoter where Tat represses BMPR2 promoter activity, truncations of the BMPR2 promoter were cloned into the pGL3 reporter plasmid and transiently transfected in the presence or absence of the Tat expression vector.
Tat-mediated repression of BMPR2 transcription decreases SMAD phosphorylation
To confirm hypothesis that Tat-mediated repression of BMPR2 decreases SMAD activation through decreased phosphorylation, U937 cells were incubated with Tat protein (50 ng/ml) or PBS for 24 h. Cells were treated with BMP2 (50 ng/ml) or PBS for 20 min, and SMAD phosphorylation levels were determined via immunoblot analysis. We observed an increase in SMAD phosphorylation in BMP2-treated cells, and following treatment with BMP2 in the presence of Tat, phosphorylation of SMAD proteins was decreased. Densitometric analysis was performed on the bands for P-SMAD and normalized to levels of TF-R, a protein that is not regulated by Tat. Results of this analysis showed an ∼2.3-fold increase in SMAD phosphorylation induced by BMP2, with almost complete inhibition of the BMP2 response by the addition of Tat.
HIV-1 infection of macrophages regulates a variety of host cell functions such as cytokine production and apoptotic signaling. Several HIV-1 proteins have been shown to be involved in this regulation, resulting in altered host cell protein expression at various levels, including post-translational trafficking of targeted proteins and transcriptional up- or down-regulation of specific genes. Numerous studies have demonstrated that HIV-1 Tat, a potent transactivator of HIV-1 transcription in both macrophages and T cells, modulates transcription from the promoters of several host genes involved in the immune response. Expression of the mannose receptor, MHC class I, MHC class II, and β2-microglobulin is down-regulated, resulting in decreased pathogen clearance and antigen presentation associated with these molecules. In the current study, we have extended these studies to Tat-mediated regulation of another macrophage surface receptor, BMPR2, which is a member of the TGF-βR superfamily. Using transient transfection assays of the BMPR2 promoter driving the luciferase reporter gene, we have shown that Tat expression in U937 cells reduced BMPR2 promoter-luciferase activity by greater than 50%. Regulation was localized to a region in the first 208 bp of the BMPR2 promoter and resulted in altered downstream signaling following ligand-receptor interaction. In addition, infection of U937 cells with a macrophage-tropic virus and treatment with recombinant Tat protein decreased endogenous BMPR2 transcript levels, suggesting that this down-regulation may occur in the setting of HIV infection.
BMPs are the natural ligands for BMPR2 and are found in most developing tissues, appearing to be principally involved in bone and cartilage formation. Most information about BMPR2 function has come from studies in the developing embryo. BMP4 and -7 colocalize in the developing lung buds, and studies examining normal and targeted misexpression of BMP4 have shown that BMP4 plays a role in embryonic lung morphogenesis. Ligation of BMPs by BMPR2 induces phosphorylation of SMAD1, -5, and -8, followed by increased transcription of the inhibitory SMAD6. In the current study, Tat not only reduces BMPR2 transcription but blocks ligand-induced signal transduction by reducing SMAD activation. A block in this pathway could have additional consequences on other pathways requiring these signaling molecules. Intracellular signaling through the SMAD pathway is initiated by BMP and TGF-β1. After binding to the respective receptors, signaling is initiated by two different subsets of receptor-bound SMADs. However, signaling from both subsets intersects at SMAD4, which facilitates nuclear translocation of BMP and TGF-β1 signaling. For example, SMAD2 and -3 are phosphorylated by TGF-β1 signaling through its receptor and converge on cytoplasmic SMAD4 in a similar manner to BMPR2 signaling. Thus, events initiated by TGF-β1 and BMP signaling compete for a limited pool of SMAD4. Perturbations of BMP signaling as a result of altered or decreased BMPR2 expression might tilt SMAD4 use toward the TGF-β1 signaling cascade.
In the current study, we have demonstrated that Tat-mediated repression of BMPR2 transcription localizes to the first 208 bp, but the mechanism involved has not yet been delineated. Tat enhancement of the rate of HIV transcriptional elongation is dependent on the interaction of Tat with a variety of host proteins including the core RNA polymerase II, TATA-binding protein associated factor (TAFII)55, TFIIH, cyclin-dependent protein kinase 7, SP1, nuclear factor of activated T cells (NFAT)1, and cyclin T1. It has been suggested that this Tat–host protein binding results in regulation of HIV transactivation and at the same time, pulls these factors away from specific host genes, altering transcription from these promoters. For example, Tat represses MHC class I transcription but does not bind directly to the promoter DNA. Inhibition appears to proceed via a direct interaction of Tat with TAFII250, a component of the general transcription factor TFIID, which is a core protein in the transcriptional initiation complex. Tat down-regulates MHC class II transcription by binding to cyclin T1, preventing the interaction of cyclin T1 with the class II transactivator CIITA. Tat interacts directly with NFAT1 in T cells, increasing NFAT1-driven transcription, leading to up-regulation of cytokines such as interleukin (IL)-2. Preliminary studies suggest that regulation of BMPR2 as well as the mannose receptor might involve a mechanism similar to MHC class II regulation, whereby Tat binding of cyclin T prevents transcriptional initiation from these promoters (R. L. Caldwell, V. L. Shepherd, manuscript in preparation).
In the current study, we have shown that Tat, expressed intracellularly in U937 cells and added extracellularly, was capable of repressing BMPR2 transcription and function. Zauli et al. reported in 1995 that Tat can be released from infected cells and act as a soluble stimulator of cell proliferation. In addition, Tat has been detected in the serum of HIV-1-infected patients at levels of 1–3 ng/ml. Tat protein includes a basic domain, which facilitates its movement across membranes and entry into the nucleus when added exogenously to cells. Effects of exogenous Tat on T cells include up-regulation of IL-2, IL-4, IL-6, and c-Fos and down-regulation of β2-microglobulin and MHC class II molecules. In macrophages, exogenous Tat transcriptionally represses mannose receptor expression and increases expression of tumor necrosis factor α (TNF-α) and IL-6. These studies expand the role for this HIV-derived protein to infected and noninfected cells, greatly expanding the potential for Tat to modulate gene expression in the infected host and contribute to a wide variety of pathologies.
The role of Tat-mediated repression of BMPR2 transcription and function in the context of HIV-infected individuals is not yet understood. It is compelling to speculate that Tat-mediated repression of BMPR2 transcription might result in HRPH, mimicking a disease that is caused by frameshift or mis-sense mutations in the BMPR2 gene, resulting in decreased BMPR2 function and/or expression. The recent finding that mutations in the BMPR2 gene can be identified in a significant percentage of patients with FPPH and SPH suggests that signaling via this receptor plays a critical role in the maintenance of normal pulmonary vascular physiology. Forty-six different germ-line mutations in the gene encoding BMPR2 have been identified, including mis-sense and frameshift mutations, which result in the loss of expression or inactivation of the receptor, but no link has been found as to how these BMPR2 mutations give rise to PH. However, the finding that arterial remodeling involves hypertrophy and proliferation of endothelial cells, smooth muscle cells, and intimal cells and that BMP2 inhibits growth of aortic vascular smooth muscle cells in vitro suggests that altered BMPR2 might lead to uncontrolled proliferation.
In the current study, we found that Tat down-regulates BMPR2 expression and signaling by ∼50%.
The role of BMPs and BMP signaling in lung disease remains at an early stage. Although clearly of direct relevance to PAH, this pathway is likely to contribute to other lung pathologies characterized by tissue remodeling, such as lung fibrosis and chronic obstructive pulmonary disease. Further exploration of the contribution of BMPs and the functional antagonism with the TGF-β pathway may reveal new targets for therapeutic intervention. Well-planned and large-scale genetic studies are required to identify additional genetic factors that increase susceptibility to PAH. Such factors may further worsen the state of BMPR-II dysfunction. Future functional studies need to identify the cell- and tissue-specific abnormalities in gene expression and cell growth/survival that are responsible for pulmonary vascular remodeling. In addition, studies are required to determine the molecular mechanism of the interaction between BMP and TGF-β pathways in lung cells. Further refinement of animal models using conditional cell-specific transgenic and knockout mice are necessary to aid in our understanding of the lung specificity of FPAH.
Reports on FPPH have suggested that this level of decreased BMPR2 expression can lead to altered lung physiology. However, we do not know if Tat-mediated reduction of BMPR2 is linked directly to the development of HRPH in HIV-infected individuals. In addition, recent studies have suggested that reductions of less than 50% in BMPR2 expression might not result in PH conditions and that mutations in other genes might be required for the PH phenotype to be expressed. For example, mutations in the TGF-βRII gene are found in tumors and atherosclerotic lesions. Reduced expression of the proapoptotic Bax gene in endothelial cells from plexiform lesions in patients with SPH could result in inhibition of apoptotic cascades. As with SPH, Tat may reduce expression of other genes in concert with BMPR2 to reach the stage of HRPH. For example, Tat up-regulates the antiapoptotic Bcl-2 gene and the cytokines TNF-α, IL-1, IL-2, monocyte chemoattractant protein-1, and TGF-β1 in macrophages. Regulation of these proteins could contribute to optimal conditions for productive HIV-1 infection and survival and also cripple the immune response to HIV and other invading pathogens. .