Subcellular enrichment of calreticulin at myoendothelial junctions (664.2)

Abstract

In the walls of resistance arteries, cellular projections from primarily endothelial cells (EC) make contact with smooth muscle cells (SMC), forming myoendothelial junctions (MEJ). The MEJ is a unique signaling microdomain responsible for multiple components of vasoreactivity. In MEJs, endoplasmic reticulum (ER) is often present; the significance of this has not been directly addressed. Using iTRAQ proteomic analysis, we discovered that calreticulin, a 50kDa calcium binding protein typically found in the ER, is highly enriched in the soluble fractions (i.e., ER) isolated from MEJs in a vascular cell co‐culture (VCCC). In the insoluble fraction (i.e., non‐ER, caveolin‐enriched lipid rafts), calreticulin was present in EC and SMC, but not MEJs. Using immuno‐TEM, mouse thoracodorsal arteries showed higher expression of calreticulin in EC (3.0 ± 0.76 beads/um2) and MEJ (2.3 ± 0.74 beads/um2) compared to SMC (0.6 ± 0.29 beads/um2). In the MEJ, beads could be identified within the ER. In order to determine a functional relevance of MEJ‐enriched calreticulin in soluble fractions, we successfully transfected EC in the VCCC with calreticulin siRNA. Next, SMC were stimulated with phenylephrine (PE) and cyclic guanosine monophosphate (cGMP) was measured. This method is known to produce a significant increase in SMC cGMP via inositol 1,4,5‐triphosphate‐based release of Ca2+ from ER at the MEJ, with subsequent activation of MEJ localized endothelial nitric oxide synthase (eNOS). The knockdown of calreticulin in EC/MEJ significantly decreased cGMP in SMC after PE stimulation. The mechanism behind this may be an interaction of calreticulin with SERCA that leads to less ER uptake of calcium, diminishing the localized calcium mobilization from the ER at the MEJ upon PE stimulation. Thus, we hypothesize that where the calreticulin is localized in cellular compartments, and not overall calreticulin expression, could be critical to MEJ function.Grant Funding Source: Supported by NHLBI 2 T32 HL007284‐36