The LPS induced pyroptosis exacerbates BMPR2 signaling deficiency to potentiate SLE-PAH.

Pulmonary arterial hypertension (PAH) is a common and fatal complication of systemic lupus erythematosus (SLE). Whether the BMP receptor deficiency found in the genetic form of PAH is also involved in SLE-PAH patients remains to be identified. In this study, we employed patient-derived samples from SLE-associated PAH (SLE-PAH) and established comparable mouse models to clarify the role of BMP signaling in the pathobiology of SLE-PAH. Firstly, serum levels of LPS and autoantibodies (auto-Abs) directed at BMP receptors were significantly increased in patients with SLE-PAH compared with control subjects, measured by ELISA. Mass cytometry was applied to compare peripheral blood leukocyte phenotype in patients prior to and after treatment with steroids, which demonstrated inflammatory cells alteration in SLE-PAH. Furthermore, BMPR2 signaling and pyroptotic factors were examined in human pulmonary arterial endothelial cells (PAECs) in response to LPS stimulation. Interleukin-8 (IL-8) and E-selectin (SELE) expressions were up-regulated in autologous BMPR2+/R899X endothelial cells and siBMPR2-interfered PAECs. A SLE-PH model was established in mice induced with pristane and hypoxia. Moreover, the combination of endothelial specific BMPR2 knockout in SLE mice exacerbated pulmonary hypertension. Pyroptotic factors including gasdermin D (GSDMD) were elevated in the lungs of SLE-PH mice, and the pyroptotic effects of serum samples isolated from SLE-PAH patients on PAECs were analyzed. BMPR2 signaling upregulator (BUR1) showed anti-pyroptotic effects in SLE-PH mice and PAECs. Our results implied that deficiencies of BMPR2 signaling and proinflammatory factors together contribute to the development of PAH in SLE.

Structure-based discovery and redesign of TGF-ß1 Elbow epitope recognition by its type-II receptor in hypertrophic scarring biotherapy.

Transforming growth factor-ß1 (TGF-ß1) signaling pathway has been implicated in the fibroblast activation of hypertrophic scarring (HS). Previously, we proposed a new biotherapeutic strategy to combat HS by disrupting the intermolecular interaction of TGF-ß1 with its cognate type-II receptor (TßR-II). Here, we further demonstrate that the binding site of TGF-ß1 to TßR-II is not overlapped with the conformational wrist epitope and linear knuckle epitope that are traditionally recognized as the functional binding sites of bone morphogenetic protein-2 (BMP-2) to its type-II receptor (BMPR-II), which can thus be regarded as a new functional site we called elbow epitope. Structural, energetic, and dynamic investigations reveal that the elbow epitope consists of two sequentially discontinuous, spatially vicinal segments Loop30-34 and Turn90-95 ; they cannot work effectively to independently interact with TßR-II. Rational redesign of the epitope is performed using an integrated in silio-in vitro method based on crystal and modeled structure data. In the procedure, the two epitope segments are split from the interface of TGF-ß1-TßR-II complex and then connected with each other in a head-to-tail manner by adding a flexible poly-(Gly)n linker between them, thus resulting in a series of combined peptides. We found that the peptide affinity reaches maximum at n = 2, which shares a consistent binding mode with the elbow epitope at native complex interface. The linker of either too long (n > 2) or too short (n < 2) cannot properly place the gap space between the two segments, thus impairing the binding compatibility of designed peptides with TßR-II active site.

Identification of a bone morphogenetic protein type 2 receptor neutralizing antibody.

OBJECTIVE: The bone morphogenetic protein (BMP) signaling pathway comprises the largest subdivision of the transforming growth factor (TGFß) superfamily. BMP signaling plays essential roles in both embryonic development and postnatal tissue homeostasis. Dysregulated BMP signaling underlies human pathologies ranging from pulmonary arterial hypertension to heterotopic ossification. Thus, understanding the basic mechanisms and regulation of BMP signaling may yield translational opportunities. Unfortunately, limited tools are available to evaluate this pathway, and genetic approaches are frequently confounded by developmental requirements or ability of pathway components to compensate for one another. Specific inhibitors for type 2 receptors are poorly represented. Thus, we sought to identify and validate an antibody that neutralizes the ligand-binding function of BMP receptor type 2 (BMPR2) extracellular domain (ECD). RESULTS: Using a modified, cell-free immunoprecipitation assay, we examined the neutralizing ability of the mouse monoclonal antibody 3F6 and found a dose-dependent inhibition of BMPR2-ECD ligand-binding. Consistent with this, 3F6 blocks endogenous BMPR2 function in the BMP-responsive cell line HEK293T. The specificity of 3F6 action was confirmed by demonstrating that this antibody has no effect on BMP-responsiveness in HEK293T cells in which BMPR2 expression is knocked-down. Our results provide important proof-of-concept data for future studies interrogating BMPR2 function.

The cooperative effect of growth and differentiation factor-9 and bone morphogenetic protein (BMP)-15 on granulosa cell function is modulated primarily through BMP receptor II.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15, GDF9B) are oocyte-derived proteins essential for the growth and function of ovarian follicles. Moreover, ovine (o) GDF9 and oBMP15 cooperate to increase both (3)H-thymidine incorporation and alpha-inhibin production and to inhibit progesterone production by rat or ovine granulosa cells. Although the receptors through which these proteins act individually have been determined, the receptor(s) involved in mediating the cooperative effects of GDF9 and BMP15 is (are) unknown. In this study, the effects of the extracellular domains of the types I and II TGFbeta receptors on (3)H-thymidine incorporation by rat granulosa cells stimulated by oGDF9 and oBMP15 were investigated. Stimulation of (3)H-thymidine incorporation was completely blocked by the BMP receptor II (BMPRII) extracellular domain but unaffected by any other type II or any type I receptor. These results suggest that the initial interaction of oGDF9 and oBMP15 is with BMPRII and that a type I receptor is either recruited or already associated with BMPRII to mediate the cooperative effects of these growth factors.

Immunoexpression of BMP-2 and BMP-4 and their receptors, BMPR-IA and BMPR-II, in ameloblastomas and adenomatoid odontogenic tumors.

OBJECTIVES: The present study evaluated the immunohistochemical expression of BMP-2 and BMP-4 and of their receptors (BMPR-IA and BMPR-II) in solid ameloblastoma (SA), unicystic ameloblastoma (UA) and adenomatoid odontogenic tumor (AOT) in order to obtain a better understanding of their role in the development and biological behavior of these tumors. DESIGN: This study analyzed these proteins in 30 cases of SA, 10 cases of UA, and 30 cases of AOT. Immunoexpression was evaluated in the parenchyma and stroma by attributing the following scores: 0, no stained cells; 1, ≤10%; 2, >10% and ≤25%; 3, >25% and ≤50%; 4, >50% and ≤75%.; 5, >75% stained cells. RESULTS: In SAs, positive correlations were observed between the stromal and parenchymal expression of BMP-2 (p<0.001) and between the stromal expression of BMP-2 and BMP-4 (p=0.020), as well as between the stromal expression of BMPR-II and BMP-4 (p=0.001) and the stromal and parenchymal expression of BMPR-II (p<0.001). In UAs, correlations were detected between the stromal and parenchymal expression of BMP-4 (p=0.035) and between the stromal expression of BMP-4 and BMPR-IA (p=0.022). In AOTs, analysis of immunoexpression in the parenchyma revealed positive correlations between all proteins. CONCLUSION: BMPs and their receptors play an important role in the differentiation and development of ameloblastomas and AOTs, but may not explain the different biological behaviors of these lesions. The positive correlation observed in AOTs might be related to the formation of mineralized material in this tumor.