The Roles of JAB1 and NOTCH1 in the Development of Cardiovascular Disease

<p>BackgroundPulmonary arterial hypertension (PAH) and Adams-Oliver syndrome (AOS) are rare vascular disorders, characterised by severe late-onset and developmental cardiac abnormalities respectively. Both of them are clinically associated in a proportion of cases. PAH is a progressive condition that is clinically characterized by sustained elevation in mean pulmonary artery pressure, through vascular remodelling with luminal obliteration of small vessels and increased vascular resistance, while AOS is characterized by congenital limb defects and scalp cutis aplasia. In a proportion of AOS-cases, notable cardiac development is also apparent. PAH may be hereditary (HPAH), idiopathic (IPAH) or associated with other conditions, for example AOS. Most of HPAH and 25% of IPAH cases are associated with heterozygous mutations of a TGF-? superfamily member transmembrane receptor type-II, known as BMPR2. The majority of mutations suggest haploinsufficiency as the molecular mechanism of disease. In addition, monoclonal proliferation and uncontrolled growth in pulmonary artery endothelial (PAEC) and smooth muscle (PASMC) cells result in perturbation and muscularization of the arterial tone. This study aims to identify the cause of a further loss of BMPR-II expression in previous immunohistochemical studies of patient’s lung sections, that cannot be explained by haploinsufficiency. The defective BMP/TGF-? signalling in PAH will be addressed as well. This study also aims to identify novel genetic determinants of AOS, along with the hithertounresolved underlying molecular defects, for the majority of affected subjects.Methods and ResultsProtein-protein interaction experimentation revealed a BMPR-II novel interacting partner, namely JAB1 protein, which through association with the kinase domain of the receptor, functions as the first identified proteasomal-associated inhibitor of BMPR-II. In addition, the linked experimental data of this study are indicative of BMPR-II degradation via the proteasomal pathway in the normal circulation. In addition, transient transfection studies demonstrated that JAB1 down-regulates BMPR-II in a dose-dependent manner. It is also confirmed that JAB1 is over-expressed during PAH progression, and proliferation studies revealed that JAB1 promotes uncontrolled cell proliferation and survival. Furthermore, functional studies supported the hypothesis that JAB1 appears to be a key regulator of BMP and TFG-? signalling pathways in PASMC cells, regulating differentially all stages of the pathways’ cascades in SMAD-dependant and SMAD-independent manners. Apart its up-stream action of promoting the proteolytic degradation of BMPR-II, RT-qPCR experimentation and further functional studies proved that it also acts down-stream, by suppressing ID1 transcription factor or by promoting ID expression via up-regulation of SMAD-dependant and SMAD-independent signalling pathways, via activation of TAK1 and p38 MAPK mitogenic cascades, thus enhancing the pro-proliferative state of healthy and/or BMPR2 mutated PASMC cells. Knock-down of JAB1 in healthy and BMPR2 mutant PASMC cells resulted in control of their proliferation, an outcome which is indicative of the essential role of JAB1 in positively regulating the cell proliferation. The anti-proliferative effect of BMP4-ligand stimulation on hyper-proliferating BMPR2mutant PASMC cells was also established, highlighting the suppressive regulatory role of BMP signalling on cell proliferation and differentiation. Regarding AOS disease, NOTCH1 mutations were found as the primary cause of AOS, in a proportion of cases of a cohort study (17%) with further cardiovascular complications. Novel identification of 10 mutations [3 frameshifts, 6 missense (EGF-like domain) and 1 nonsense] of the NOTCH1 gene in AOS patients, along with a significant reduction of NOTCH1 expression in leukocyte-derived RNA from subjects, suggested that NOTCH1 plays a key role in the AOS pathogenesis. Transient transfections of mutagenized NOTCH1 missense constructs also revealed significant reduction in gene expression. Likewise, assessment of NOTCH1 target genes HEY1 and HES1 expression via RT-qPCR studies, verified the dysregulation of the NOTCH1 canonical signalling pathway in AOS-patients. Lastly, functional studies demonstrated the cross talk of BMP and NOTCH1 signalling as a synergistic effect in AOS pathology, underlined by forms of non-canonical signalling, potentiating the suppressive BMP cell signalling effect in AOS-cases of dysfunctional NOTCH1 signalling and further cardiovascular defects.ConclusionsThese findings manage to provide further insight into JAB1 mediated BMPR-II down-regulation and degradation via the proteasomal pathway, and determination of JAB1 dysregulation and role in the aggressive mitogenic proliferation potential, through reduction of SMAD signalling and consequently p38 MAPK and TAK1 activation, observed in PAH cells. This study highlights the significance of this molecule in the vasculature and sets it as a potential novel target in PAH therapy, and for drugs against PASMC cells in pro-proliferating state harbouring or not BMPR2 PAH-associated mutations. Finally, it is established that haploinsufficiency of the NOTCH1 receptor is a primary cause of AOS, while solidifies NOTCH1 as an important genetic factor in AOS with associated cardiovascular complications. Further X-ray crystallography studies of the identified NOTCH1 missense mutations of the EGF-like domain will also elucidate the extend of the structural perturbation of the receptor and subsequent impairment of the signalling cascade.</p>