Tissue Specific Expression of Connexins 26, 37, 40, and 43 in the Mouse by RT-PCR Analysis
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Brian P. Fox
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Introduction
Abstract
Connexins are a family of proteins involved in the formation of gap junctions. Gap junctions are involved in cell to cell adhesion as well as in cell to cell communication. There are 14 different connexin genes which come together to form either homomeric or heteromeric hemichannels which then pair up to form transmembrane channels between adjacent cells. The tissue specific expression of connexins26, 37, 40, and 43 in mouse heart, liver and lung was studied using RT-PCR analysis and Sanger sequencing. All four connexins studied were present in all three tissues with the exception of connexin26 which was absent from the heart.
Introduction
Connexins are a family of proteins contributing to the formation of gap junctions. Gap junctions are transmembrane channels that contribute to cell adhesion, as well as metabolically coupling adjacent cells by permitting the passage of ions and small molecules such as cAMP and Ca2+[1,2]. To date, there have been 14 identified connexin proteins, all part of the connexin multigene family. These connexins vary in molecular weight from 26 KDa to 57 KDa. A complete gap junction consists of two hemichannels called connexons from adjacent cells connecting to form a channel spanning the plasma membrane of both cells. Each connexon consists of 6 connexins, and is either homomeric, if all six connexins are the same, or heteromeric, if more than one type of connexin is present[3]. Junctions made of different connexins have different permeability’s and regulatory properties[4].
The expression of connexins has been shown to vary between tissues. Some connexins are expressed in a variety of tissues (e.g. Cx-37, Cx-40, and Cx-43), while others are more sparsely expressed (e.g. Cx 26)[5]. Cx-37, Cx-40, and Cx-43 are expressed in endothelial cells, including the lining of blood vessels and may explain their presence in many organs[6,7]. Commonly the expression of connexins in varying tissues has been determined by immunohistochemistry, although analysis by reverse transcription-polymerase chain reaction (RT-PCR) for many tissues and connexins has been performed[7-10]. The benefit of immunohistochemistry is that the relative levels of a specific connexin between tissues can be easily quantified by comparing the level of connexin specific staining. It is difficult to determine if the channels are homomeric or heteromeric, but the overall amount of a connexin can be quantified. Although the relative amounts of a particular connexin in a tissue can also be determined with RT-PCR by varying the amounts of mRNA template used for first strand synthesis, its true benefit is its ability to test for either the presence or absence of a specific mRNA, in this case connexin mRNA’s, even if present in only small amounts.
The variability involved in connexon composition allows for a great deal of diversity in gap junction formation. Each gap junction appears to confer some specificity for what type of molecules pass through it, either based on charge or size of the molecule[9]. Based on that specificity, it seems likely that even small amounts of a particular gap junction with a unique composition of connexins could be important for the movement of a particular metabolite or set of metabolites. Identifying what connexins are present in a particular tissue, even if only in small amounts could thus be crucial for understanding their roles in cell communication as well as cell adhesion. For this reason, the presence of Cx-26, Cx-37, Cx-40, and Cx-43 in the lung, liver and heart of a mouse were tested for by RT-PCR
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Figures
Figure 1-Total RNA isolated from mouse lung (lane 1), liver (lane 2), and heart (lane 3). A 100 bp ladder precedes lane 1.
Figure 2-DNased treated RNA
Figure 3-PCR products from cDNA created from lung(lanes 1-4), liver(lanes 5-8), and heart(lanes 9-12) using primers specific for Cx26(lanes 1,5,and 9), Cx37(lanes 2,6,and 10), Cx40(lanes 3,7,and 11), and Cx43(lanes 4,8,and 12). A 100 bp ladder precedes lane 1.
Figure 4-PCR test for presence of genomic DNA. Primers spanning a 1kb intron were used to test for the presence of genomic DNA in the lung (lane 1), liver (lane 2), and heart (lane 3) cDNA template. A 100 bp ladder precedes lane 1.
Results
RNA extraction from the three tissues: heart, liver, and lung yielded good products (Figure 1). There did appear to be some DNA contamination, as evidenced by the presence of a band at the top of the gel. These bands are not easily identified in the reproduction of the picture, but were clearly visible under UV light and in the picture of the gel. The two bright bands correspond to the 28S and 18S ribosomal subunits. After Dnase treatment of the samples, the band of DNA contamination has been lost, with only a slight decrease in the intensity of the ribosomal subunit bands. (Figure 2).
Figure 3 represents the tissue specific expression of the 4 connexins in question (Cx-26, Cx-37, Cx-40, and Cx-43). Connexins 37, 40, and 43 have been amplified from the mRNA’s from each of the three tissues: heart, liver, and lung, while Cx-26 is present in the liver and the lung, but absent from the heart. Cx-26 from both the liver and the lung showed a weaker band than the other 3 connexins, and also a higher degree of non-specific binding not visible in this picture because the “Gene-Cleaned” PCR product was used to run on the agarose gel.
The bands present were amplified from cDNA synthesized from the mRNA present in the tissues, not genomic DNA(Figure 4). The PCR products using primers BFCx37IF and BFCx37IR which spanned intron 1 in Cx-37 were around 200 bp as would be expected from amplification of the portion of Cx-37 lacking an intron. If the intron was present, the band would have been 1.2kb, and the amplification would have been from genomic DNA, not cDNA.
Sequencing confirms the bands amplified correspond to the connexin gene targeted by the connexin specific primers(Data not shown). Each purified PCR product used as a template for sequencing showed high homology (>94% to the mouse connexin gene it was thought to be). The sequences recorded were compared to regions of the connexin gene that were significantly different from other connexin genes, thus confirming the presence of the varying connexin genes in the heart, liver, and lung.
Discussion
The results of these experiments confirms the presence of Cx-37, Cx-40, and Cx-43 in all three tissues. As stated before, these three connexins are thought to be expressed in the endothelial lining of blood vessels, so there presence in all three tissues is not surprising. More intricate dissections would have to be performed to remove the blood vessels and study the expression in the tissue cells alone. The absence of Cx-26 in the heart also confirms previous studies which have shown it is not expressed in this tissue[5]. It would be interesting to measure the relative amounts of these connexins using dilution’s of the template for PCR amplification and comparing the strength of the bands from each tissue. Expression of mutated connexins or their absence in a tissue have been correlated to many diseases including deafness when Cx-26 is misexpressed in the ear, as well as abnormal development of the heart when Cx-37, Cx-40, or Cx-43 are misexpressed (mutated or absent)[11-15]. It may turn out that particular combinations of connexons coming together to form gap junction are responsible for the movement of particular metabolites, so the identification of specific gap junctions, even if present in only small amounts appears to be important. If this is the case, we will need to determine what connexins are expressed in tissues and in what amounts. Once this has been determined, it may be possible to identify what molecules are permitted to pass through specific channels and this could be important in learning the exact causes for connexin-related disorders. It is the identification of connexin mRNA’s present in only small amounts which makes this RT-PCR technique so powerful in this field.
Full Paper
Acknowledgments
Acknowledgments
Sincere thanks go out to Sabrina Volpi, Ira Daly, and Rocco Coli for their patience and guidance throughout this project. Without their help, this project would not have been possible. Thanks also to Dr. Berish Y. Rubin for the use of the lab and support throughout the semester as well as for providing me with this opportunity to pursue this individual project. A special thanks to Iman for valuable discussions and expertise which were provided during my time in the lab.
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