Protocadherins (Pcdhs) are a large family of cadherin-related molecules. They play a role in cell adhesion, cellular interactions, and development of the central nervous system. However, their expression and role in endothelial cells has not yet been characterized. Here, we examined the expression of selected clustered Pcdhs in endothelial cells from several vascular beds. We analyzed human and mouse brain microvascular endothelial cell (BMEC) lines and primary cells, mouse myocardial microvascular endothelial cell line, and human umbilical vein endothelial cells. We examined the mRNA and protein expression of selected Pcdhs using RT-PCR, Western blot, and immunostaining. A strong mRNA expression of Pcdhs was observed in all endothelial cells tested. At the protein level, Pcdhs-gamma were detected using an antibody against the conserved C-terminal domain of Pcdhs-gamma or an antibody against PcdhgC3. Deletion of highly expressed PcdhgC3 led to differences in the tight junction protein expression and mRNA expression of Wnt/mTOR (mechanistic target of rapamycin) pathway genes as well as lower transendothelial electrical resistance. Staining of PcdhgC3 showed diffused cytoplasmic localization in mouse BMEC. Our results suggest that Pcdhs may play a critical role in the barrier-stabilizing pathways at the blood-brain barrier.

Inflammation of the central nervous system (CNS) is associated with diseases such as multiple sclerosis, stroke and neurodegenerative diseases. Compromised integrity of the blood-brain barrier (BBB) and increased migration of immune cells into the CNS are the main characteristics of brain inflammation. Clustered protocadherins (Pcdhs) belong to a large family of cadherin-related molecules. Pcdhs are highly expressed in the CNS in neurons, astrocytes, pericytes and epithelial cells of the choroid plexus and, as we have recently demonstrated, in brain microvascular endothelial cells (BMECs). Knockout of a member of the Pcdh subfamily, PcdhgC3, resulted in significant changes in the barrier integrity of BMECs. Here we characterized the endothelial PcdhgC3 knockout (KO) cells using paracellular permeability measurements, proliferation assay, wound healing assay, inhibition of signaling pathways, oxygen/glucose deprivation (OGD) and a pro-inflammatory cytokine tumor necrosis factor alpha (TNFα) treatment. PcdhgC3 KO showed an increased paracellular permeability, a faster proliferation rate, an altered expression of efflux pumps, transporters, cellular receptors, signaling and inflammatory molecules. Serum starvation led to significantly higher phosphorylation of extracellular signal-regulated kinases (Erk) in KO cells, while no changes in phosphorylated Akt kinase levels were found. PcdhgC3 KO cells migrated faster in the wound healing assay and this migration was significantly inhibited by respective inhibitors of the MAPK-, β-catenin/Wnt-, mTOR- signaling pathways (SL327, XAV939, or Torin 2). PcdhgC3 KO cells responded stronger to OGD and TNFα by significantly higher induction of interleukin 6 mRNA than wild type cells. These results suggest that PcdhgC3 is involved in the regulation of major signaling pathways and the inflammatory response of BMECs.

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