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Calreticulin localized to the myoendothelial junction mediates heterocellular communication in resistance arteries
Author(s) -
Biwer Lauren A,
Good Miranda E,
Hong Kwangseok,
Patel Rahul,
Agrawal Neha,
LooftWilson Robin,
Sonkusare Swapnil,
Isakson Brant E
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.681.12
Subject(s) - calreticulin , endoplasmic reticulum , microbiology and biotechnology , biology , calcium binding protein , unfolded protein response , kdel , calcium signaling , vascular smooth muscle , ryanodine receptor , calcium , signal transduction , medicine , endocrinology , smooth muscle , golgi apparatus
Regulated heterocellular communication depends on bidirectional signaling that occurs at myoendothelial junctions (MEJs), which are composed of endothelial cell (EC) extensions that make direct cytoplasmic contact with smooth muscle cells (SMC). The MEJ is the anatomical hallmark of resistance arteries and is a key signaling microdomain with endoplasmic reticulum (ER) as we demonstrate via expression of the ER retention signal –KDEL, and ER tracker in a live artery, both of which localize to holes in the internal elastic lamina. Presumably, the expression of ER in the MEJ could facilitate localized Ca 2+ events at MEJs generated in response to second messengers from EC agonists or smooth muscle (SMC) agonists (by movement of IP 3 or Ca 2+ through gap junctions to the EC extensions composing MEJs). To determine what proteins may regulate the spatially localized Ca 2+ events at the MEJ, we used an iTRAQ proteomic screen on in vitro MEJs and found significant enrichment of calreticulin. Calreticulin is a calcium binding protein that has been shown to bind ~50% of ER calcium. We confirmed the presence of calreticulin at MEJs using third‐order mesenteric arterioles. Thus, we hypothesized that deletion of EC calreticulin could alter Ca 2+ events at the MEJ that were generated after application of EC or SMC agonists. For this reason we created an EC specific calreticulin conditional knockout mouse ( Calreticulin fl/fl / CDH 5‐CreER T2+ ). Initial experiments were performed in mice between 10–20 weeks old. In these experiments, carbachol (CCH; EC specific) or phenylephrine (PE; SMC specific) was added to third order mesenteric arteries and the Ca 2+ events were quantified. The control mice had a robust increase in Ca 2+ events at MEJs after PE stimulation, but Calreticulin fl/fl / CDH5 ‐CreER T2+ mice had a significantly reduced number of Ca 2+ events. Furthermore, Calreticulin fl/fl / CDH5 ‐CreER T2+ mice had significantly enhanced constriction to PE, demonstrating the importance of calreticulin at the MEJ in the PE response. Conversely, CCH stimulation showed no differences in Ca 2+ events and no difference in dilation to CCH in the Calreticulin fl/fl / CDH5 ‐CreER T2+ mouse. This polarized response in Ca 2+ events at MEJs in Calreticulin fl/fl / CDH5 ‐CreER T2+ endothelium was lost in mice aged >75 weeks. These results demonstrate: 1) the enrichment of calreticulin to MEJs, 2) polarized activation of EC Ca 2+ events is based upon whether the agonist is EC (apical) or SMC (MEJ) in origin and 3) aging alters how calreticulin polarizes Ca 2+ events after agonist stimulation. Overall, we believe EC calreticulin can regulate Ca 2+ events generated in responses to heterocellular communication. Support or Funding Information National Institutes of Health HL088554, HL120840, T32 HL007284, and American Heart Association 14PRE20420024