Calcium gives the final touch to the formation of functional endothelial barriers

Epithelial tissues provide the body’s first line of protection from physical, chemical, and biological wear and tear. The endothelia establish the paracellular barrier to prevent plasma proteins and cells in the blood from entering tissues. These cells act as gatekeepers of the body controlling permeability and allowing selective transfer of materials across a physical barrier. Any substance in the environment must overcome the epithelium. Toxic molecules, cellular debris, and microorganisms in the blood stream must bypass the tight junctions (TJ) of the endothelium. It is well known that calcium has a role in forming and organizing cellular structures and helps regulate physiological functions. The endothelial cells exhibit polarity with differences in structure and function between the exposed or apical facing surface of the cell and the basal surface close to the underlying body structures. This polarization helps control many activities of cells. The apical junctional complex (AJC) consists of a large number of proteins that are potentially interconnected into a very complex protein network that is located at the apex of the lateral membrane of polarized endothelial cells and comprises a highly organized structure involved in regulating cell-cell adhesion, paracellular permeability, and cell polarity. AJC contains two important subdomains, the TJ and adherens junction (AJ). This AJC on the endothelial cells plays a vital role tissue homeostasis.

In a new research published in FEBS Letters, researchers from Brigham Young University Christopher Mendoza, Sai Harsha Nagidi, Kjetil Collett, and Jacob Mckell, led by Professor Dario Mizrachi, conducted a study to understand how TJ and AJ are assembled to form the AJC. To date, the underlying mechanism of the formation of AJC through a combination of TJ and AJ remains unclear. Better understanding of the interaction would enhance our understanding of barrier formation in endothelial and epithelial cell physiology, improve drug delivery, and discover new therapeutic drug targets. For the purpose of the study, researchers synthesized TJ proteins, claudin (CLDN), occludin (OCLN), and junctional adhesion molecules (JAMs) as maltose-binding protein fusion. To understand the role of JAMs, they prepared epithelial cadherin (E-CAD) soluble domains fused C-terminal to MBP. E-CAD are calcium-dependent adhesion molecules that form the AJ.

The author’s main objective was to answer a basic question, already answered for E-CAD, and based on the fact that extracellular calcium concentration are 1,000 times higher than inside the cell. Can calcium influence TJ components and do TJ components interact due to calcium? Additionally, do TJ and AJ interact due to calcium? To answer these questions, Professor Dario Mizrachi and his colleagues worked with recombinant tools to express functional versions of these membrane proteins. A key element in this work is the use of functional proteins to examine the effects of calcium. Until now experiments in this research area has been relegated to cellular and tissue-based experimentation. With these proteins uniquely prepared, they used a variety of elegant analytical experiments such as size-exclusion chromatography (SEC) and Circular Dichroism spectrometry, and Surface Plasmon resonance to address the questions. They reported that the self-binding affinity of JAMs increased 7-fold from calcium-free conditions in presence of calcium. Similarly, homotypic interaction of CLDNs increased when exposed to calcium. However, in the case of OCLN, this value decreased in the presence of calcium. This demonstrated that calcium affects the assembly of the TJ, AJ and the AJC by controlling protein-protein affinities of some of these membrane proteins.

In a statement to Medicine Innovates Professor Mizrachi said: “The cell adhesion properties of the TJ and AJ proteins is fully realized on the cell surface, where the environment favors cell-cell interactions and builds up cell integrity by forming functional barriers”

To demonstrate the calcium depletion/repletion (switch) hypothesis, the authors showed that calcium plays an important role in the interaction between TJ and AJ, ensuring their coordinated assembly into the AJC. The authors found that it is the high concentration of calcium outside the cells that acts as a switch for the assembly of these protein complexes, with JAMs acting as interface molecule between the TJ and AJ, leading to the formation of an active AJC.

In summary, Professor Dario Mizrachi and colleagues using protein engineering and biophysical studies have greatly expanded our knowledge of TJ formation and how calcium affects the initiation of endothelial cells polarity and functional barriers, vital for homeostasis. However, there is still much to be learned about these processes and their role in disease. Moreover, the methods used in this study provide an alternative new tool for the classical cell-based approach into examining how TJs protein complexes are assembled and regulated. By understanding how the AJCs are assembled in the cells, researchers can find better ways of improving tissue integrity, improving drug delivery, and preventing various chronic disorders, including cancer.