Gap Junction

The Lampe laboratory performs studies to find blood tests or protein biomarkers that can indicate if and where a person has cancer and if it is present how to best treat it. Specifically, we are working to find early detection, recurrence or response biomarkers of colon, breast, pancreas and lung cancer. Useful biomarkers can allow doctors to find and treat cancer earlier and better saving lives, reducing costs and allowing for “personalized or precision medicine” where the treatment is specific for each person’s disease. Although in the past we have used a variety of mass spectrometry methods, currently our primary approach is to utilize high density antibody arrays to determine proteomic, glycoproteomic and auto-autoantibody markers of disease.

We are especially interested in markers where the level of a protein, the level of its glycosylation or whether autoantibodies are produced to it can yield multi-dimensional information on each protein. We consider specific proteins that show consistent cancer-specific changes in 2 or 3 of these measurements to be “hybrid markers”. We hypothesize these markers will suffer less variation between different individuals since one component can act to “standardize” the other measurement. Our approach to biomarker discovery is unique in several ways. Our discovery arrays contain over 3000 antibodies printed in triplicate giving us reliable and highly consistent data. The fact that we assay proteomic, glycomic and autoantibody changes gives us broad coverage of potential biomarkers. We can screen hundreds of samples in a week reducing false positives. For early detection, we have utilized large pre-diagnostic sample sets from screening cohorts (WHI, CHS, PLCO) reducing the chance that potential early detection biomarkers are not simply related to inflammation or disease burden. We validate in similar sized sample sets and the high denisty of the arrays allow us to retain hundreds of candidate biomarkers. Thus, we do not spend the time to develop individual assays until biomarkers have passed multiple validation steps and their utility is more clear reducing costs and prioritizing effort.  

Cx43 Phosphorylation

The C-terminal tail of Cx43 contains numerous regulatory sites. Our focus in studying gap junction biology is to understand which kinases and signaling pathways regulate phosphorylation of Cx43. One interesting characteristic of Cx43 is that phosphorylation can change the migration of protein on SDS-PAGE. As a result, Cx43 typically migrates as three or more bands, and we have made great progress in understanding which phosphorylation sites cause the migration shift. We have also discovered that Cx43 phosphorylation at serine 365 serves as a ‘gatekeeper’ role by preventing downregulation of gap junctional communication by subsequent PKC phosphorylation at serine 368 cannot be phosphorylated. Src activation leads to Src, MAPK and PKC phosphorylation of Cx43 at 6 serines and 2 tyrosines also decreasing gap junction function. Phosphorylation at CK1 or PKA-related sites cause changes in Cx43 structure that increase GJ assembly.  

Cx43/pS368 staining of human wounds
Cx43/pS368 staining of human wounds

Epidermal Wound Healing

Wound healing requires concerted action of various signals to activate cells to migrate over the wound bed and in a different zone to divide to replace the damaged ones. Cx43 must be regulated during this process, or wounds do not heal properly. In healthy skin, Cx43 is localized to differentiated cells of the epidermis and excluded from the basal cell layer. In wounded skin, Cx43 is sequentially phosphorylated on sites that change communication and then lead to gap junction degradation, such that total Cx43 protein is low in damaged cells and those adjacent to them. Then after 24 hours from the time of wounding, cells in the basal layer express Cx43 phosphorylated on serine 368 crating a unique communication compartment. We are working to understand how these various phosphorylation events affect the different cell processes required for wound healing. 

Cardiac ischemia
Cx43 phosphorylation

Cardiac Ischemia

Ischemia (i.e., lack of O2) injury and subsequent restoration of oxygenated blood flow in the heart results in tissue remodeling termed ischemia reperfusion injury. Remarkably, this damage can be limited if hearts are preconditioned, that is, if they are first exposed to short bouts of ischemia prior to a longer duration of ischemia and reperfusion. One hallmark of the remodeling process is the change in localization of Cx43 from the intercalated disk to the lateral edges of ventricular myocytes. We have shown that in healthy hearts, virtually all of the Cx43 in ventricular tissue is phosphorylated on specific sites, but after ischemia, sites associated with gap junction assembly (i.e., S365 and S325, 328, 330) are de-phosphorylated, while other sites such as S373 that are associated with increased gap junction size (see Figure) and others with subsequent gap junction turnover and closure are increased. Since recent work has shown that hearts lacking one allele of Cx43 cannot be precondition we are investigating the role Cx43 phosphorylation plays in cardioprotection. 

Alterations during skin carcinogenesis
Cx43-p368 Alterations during skin carcinogenesis


Based on the observations that primary tumors express decreased levels of connexins and that re-expressing connexins in tumor cell lines limits their growth, connexins and gap junctions are considered to be tumor suppressors.  However, the role of connexins in tumorigenesis is much more complicated because connexins are expressed in several different cell types, and their roles are likely to depend on tissue microenvironment and the cell type of interest.  For example, during skin carcinogenesis, we find that levels are actually increased in hyperplastic lesions but Cx43 is reduced in papillomas and becomes mislocalized in squamous cell carcinoma as cancer progresses.  We are interested in how Cx43 phosphorylation contributes to tumorigenesis and are currently investigating the effects of Cx43 expression and the role of specific phosphorylation sites in the development of pancreatic ductal adenocarcinoma (PDA).

Cx43 in the Eye
Cx43 in the Eye

Cx43 Expression and Phosphorylation in the Eye

Cx43 is abundantly expressed in the developing eye in the lens, cornea and retina. Furthermore, the expression and phosphorylation of Cx43 changes during development. Specifically, we know the phosphorylation of Cx43 at the neural retina/pigmented retina border is phosphorylated very specifically at serine 368. Since this phosphorylation event causes the channel properties to change, molecule exchange between these two cell types is presumably different. 


Funding: These studies are currently funded by a grant from NIH (GM55632).