Recent molecular and pharmacological studies performed on non-cardiac tissues suggest that tumor endothelial marker-8 (TEM-8), also known as anthrax toxin receptor 1 (ANTXR1) and capillary morphogenesis gene-2 (CMG-2), known as anthrax toxin receptor 2 (ANTXR2) regulate survival, proliferation, cell shape, as well as polarity, adhesion, migration and differentiation. However, downstream signaling mechanisms and function of these molecules have not been studied. Recent studies indicate that activation of protein kinase B (Akt) and c-Jun N-terminal kinases (JNK) have been associated with hypertrophic growth, as well as regulation of contractility in cardiac myocytes (CM).
In this study we’ve investigated the role of ANTXR1 and ANTXR2 receptors in intracellular pathways of contractility regulation in neonatal rat ventricular myocytes (NRVM).
Primary culture of NRVM was used to determine the effects of ANTXR1/ANTXR2 activation on Akt and JNK phosphorylation by Western blot analysis. Flow cytometry and immunostaining of alive CM were used to determine expression levels and distribution of anthrax receptors throughout the cells. Furin-activated form of anthrax protective antigen (PA
63
) was used in order to selectively stimulate ANTXR1/ANTXR2 receptors and get understanding of downstream signaling mechanisms coupled to them. The treatments with angiotensin II, type1 receptor (AT
1
) inhibitors were used in order to separate responses of distinct regulatory pathways of contractility. All treatments were done at variable time courses started in seconds and finished in half of hour to determine a sequence of phosphorylation reactions going from cellular membrane to deep inside of cell. As a result, abundant expression of both receptors was observed in cardiac myocytes. Activation of one phospho-site of focal adhesion kinase (FAK
861
) was started in 15 sec with subsequent phosphorylation of JNK and Akt
473
in 5 and 10 minutes. Involvement of new molecules was observed in cascade chain reaction of contractility regulation.
In conclusion, ANTXR1/ANTXR2 were demonstrated to couple to mechano-sensor molecules: FAK, Akt and JNK, as well as interact with the AT
1
receptors to mediate downstream signaling events responsible for regulation of contractility.
Endosomal recycling regulates anthrax toxin receptor 1/tumor endothelial marker 8-dependent cell spreading