Cutaneous and hepatic vascular lesions due to a recurrent somatic GJA4 mutation reveal a pathway for vascular malformation

Abstract

The term "cavernous hemangioma" has been used to describe vascular anomalies with histology featuring dilated vascular spaces, vessel walls consisting mainly of fibrous stromal bands lined by a layer of flattened endothelial cells, and an irregular outer rim of interrupted smooth muscle cells. Hepatic hemangiomas (HHs) and cutaneous venous malformations (VMs) share this histologic pattern, and we examined lesions in both tissues to identify genetic drivers. Paired whole-exome sequencing (WES) of lesional tissue and normal liver in HH subjects revealed a recurrent GJA4 c.121G>T (p.Gly41Cys) somatic mutation in four of five unrelated individuals, and targeted sequencing in paired tissue from 9 additional HH individuals identified the same mutation in 8. In cutaneous lesions, paired targeted sequencing in 5 VMs and normal epidermis found the same GJA4 c.121G>T (p.Gly41Cys) somatic mutation in three. GJA4 encodes gap junction protein alpha 4, also called connexin 37 (Cx37), and the p.Gly41Cys mutation falls within the first transmembrane domain at a residue highly conserved among vertebrates. We interrogated the impact of the Cx37 mutant via lentiviral transduction of primary human endothelial cells. We found that the mutant induced changes in cell morphology and activated serum/glucocorticoid-regulated kinase 1 (SGK1), a serine/threonine kinase known to regulate cell proliferation and apoptosis, via non-canonical activation. Treatment with spironolactone, an inhibitor of angiogenesis, suppressed mutant SGK1 activation and reversed changes in cell morphology. These findings identify a recurrent somatic GJA4 c.121G>T mutation as a driver of hepatic and cutaneous VMs, revealing a new pathway for vascular anomalies, with spironolactone a potential pathogenesis-based therapy.

Figure 1

Shared features of HHs and cutaneous venous malformations (A) A 16 × 11 × 16 cm vascular mass (white dashed lines and white arrow) in the right liver lobe consistent with HH found in VASC102. (B) Histology of this lesion demonstrates dilated vessels filled with red blood cells. Scale bar, 500 μm. (C) Immunohistochemistry (IHC) demonstrating CD31 immunoreactivity in endothelial cells lining vessels confirms that these are vascular lesions. Scale bar, 75 μm. (D and E) Cutaneous venous malformations show similar histology with dilated vascular spaces and vessel walls consisting mainly of fibrous stromal bands lined by a single layer of flattened endothelial cells. Scale bar, 500 μm.

Figure 2

Cx37-Gly41Cys expression induces morphological changes in HUVECs and downregulates expression of proteins comprising the autophagy initiation complex in endothelial cells (A) Overexpression of HA-tagged Cx37-WT and Cx37-Gly41Cys was verified via western blotting. Actin is blotted as a loading control. (B) HUVECs expressing Cx37-WT demonstrate a cobblestone morphology. Scale bar, 150 μm. (C) HUVECs expressing Cx37-Gly41Cys exhibit large cytoplasmic vacuoles and spindled morphology. Scale bar, 150 μm. (D) Expression of Cx37-Gly41Cys leads to increased levels of the autophagosome markers LC3A/B (LC3A/B). Compared to Cx37-WT, Cx37-Gly41Cys leads to decreased expression of proteins comprising the autophagy initiation complex. Taken together, these data suggest that Cx37-Gly41Cys inhibits autophagosome initiation or processing. Actin is blotted as a loading control.

Figure 3

Cx37-Gly41Cys expression activates SGK1 via a non-canonical pathway (A) HUVECs transduced with Cx37-G41C express a higher molecular weight phosphorylated form of SGK1, with expression of SGK1-Ser78 seen exclusively in Cx37-Gly41Cys cells. Cx37-Gly41Cys does not alter MAPK expression. Cx37-WT leads to decreased Akt-Ser473 expression, whereas Cx37-Gly41Cys expression results in almost complete abrogation. Actin is blotted as a loading control. (B) Gradient induction of Cx37-Gly41Cys expression with doxycycline leads to dose-dependent increases in phosphorylation of SGK1 at serine 78 and an increase in total SGK1 expression. In the absence of Cx37-Gly41Cys expression, SGK1 runs as a doublet on SDS-PAGE (lane 1) but migrates as a single band when expression of the mutant protein is induced. These data indicate that Cx37-Gly41Cys leads to SGK1 activation via serine 78 phosphorylation. Actin is blotted as a loading control. (C) Immunohistochemical staining reveals absence of SGK1-Ser78 in vasculature of normal liver tissue and strong SGK1-Ser78 expression in HH tissue. Scale bars, 75 μm.

Figure 4

Spironolactone inhibits and reverses SGK1 non-canonical activation and morphological changes in HUVECS expressing Cx37-Gly41Cys (A) SGK1 activation via phosphorylation at serine 78 is seen in cells overexpressing Cx37-Gly41Cys in the absence of spironolactone but is not present with addition of spironolactone at the time of induction of Cx37-Gly41Cys expression or 10 h after. Lysates were prepared at 18 h. (B and C) Cytoplasmic vacuoles are seen in Cx37-Gly41Cys HUVECs that are not treated with spironolactone (B), but addition of spironolactone reverses this phenotype by 14 h after treatment (C). Images were taken at 24 h after induction of Cx37-Gly41Cys expression. Scale bars, 150 μm.