MxA Is a Novel Regulator of Endosome-Associated Transcriptional Signaling by Bone Morphogenetic Proteins 4 and 9 (BMP4 and BMP9)

Abstract

There is confusion about the role that IFN-α plays in the pathogenesis of pulmonary arterial hypertension (PAH) with different investigators reporting a causative or a protective role. There is now clear evidence in PAH pathogenesis for the involvement of BMP4 and BMP9 signaling, and its disruption by mutations in BMPR2. In the present study, we investigated MxA, an IFN-α-inducible cytoplasmic dynamin-family GTPase for effects on BMP4/9 signaling, including in the presence of PAH-disease-associated mutants of BMPR2. In human pulmonary arterial endothelial cells (HPAECs), IFN-α-induced endogenous as well as exogenously expressed MxA was associated with endosomes that aligned alongside microtubules and tubules of the endoplasmic reticulum (ER). Moreover, IFN-α and MxA stimulated basal and BMP4/9 signaling to a Smad1/5/8-responsive pBRE-Luc reporter. In HEK293T cells, immunoelectron microscopy confirmed the association of MxA with endosomes, and immunofluorescence methods showed these to be positive for early endosome markers (early endosomal antigen 1, clathrin light chain and Rab5) and RSmad1/5/8. Functionally, using different genetic and inhibitor approaches, we observed that clathrin-mediated endocytosis enhanced and caveolin-mediated endocytosis inhibited the transcriptional response to BMP4 and BMP9. MxA produced a further 3-4-fold enhancement of the BMP-induced response in a clathrin-endocytosis dependent manner. The microtubule inhibitor nocodazole and stabilizer paclitaxel respectively attenuated and enhanced the effect of MxA, implicating microtubule integrity in this process. MxA enhanced BMP-induced signaling in the presence of wild-type BMPR2, and partially rescued signaling from some PAH-disease-associated BMPR2 mutants. Taken together, the data identify MxA as a novel stimulator of BMP4 and BMP9 transcriptional signaling, and suggest it to be a candidate IFN-α-inducible mechanism that might have a protective role against development of PAH and other vascular diseases.

Human antiviral protein MxA forms novel metastable membrane-less cytoplasmic condensates exhibiting rapid reversible “crowding”-driven phase transitions

“…2C). As expected from our previous studies (22, 27), this MxA reticulum was distinct from the standard endoplasmic reticulum when tested using an antibody to the ER structural protein RTN4 (28–31) (Fig. 2D).…”

“…A noteworthy feature of the appearance of cytoplasmic MxA structures illustrated in the literature (15-20, 23-25) as well as in our hands (22, 27) was their marked phenotypic variation. Figs.…”

“…S8). As with our previous data (22, 27), the present data did not allow us to localize MxA significantly to any portion of the ER (also see below Fig. S12 for double-label immuno-EM studies).…”

“…However, none of the previous studies characterized MxA localization using authentic markers of the ER such as known structural or luminal ER proteins. In our recent studies using reticulon-4 (RTN4), a structural protein of the mammalian ER, as a marker we were unable to localize MxA to the ER (22, 27). We were also puzzled by the diversity of size and shape of cytoplasmic MxA structures in the published MxA literature (15-20, 23-25) which did not appear to match structural features of the ER (22, 27-32).…”

The large GTPase Mx1 binds Kif5B for cargo transport along microtubules

“…It thus seems that Mx1 accumulates on contact sites between the ER and the endosomal system. In line with this idea, Mx1 or its orthologue MxA has been found on the smooth ER and on endosomal membranes . The question is what role Mx proteins play on these membranes?…”