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Vertebratae homologues of Innexins May Form Gap Junctions Important in Ischemia and Tumorigenesis

This work has been started in collaboration with Dr.Yury Panchin (Institute of Problems of Information Transmission, Russian Academy of Science)


The mammalian pannexin family is homologous to the invertebrate innexin gap junction proteins.

Baranova A, Ivanov D, Petrash N, Pestova A, Skoblov M, Kelmanson I, Shagin D, Nazarenko S, Geraymovych E, Litvin O, Tiunova A, Born TL, Usman N, Staroverov D, Lukyanov S, Panchin Y.

In 2000-2002, we have cloned the genes PANX1, PANX2 and PANX3, encoding putative gap junction proteins homologous to invertebrate innexins, which constitute a new family of mammalian proteins called pannexins. Phylogenetic analysis revealed that pannexins are highly conserved in worms, mollusks, insects and mammals, pointing to their important function. Both innexins and pannexins are predicted to have four transmembrane regions, two extracellular loops, one intracellular loop and intracellular N and C termini. Both the human and mouse genomes contain three pannexin-encoding genes. Mammalian pannexins PANX1 and PANX3 are closely related, with PANX2 more distant.

This study has been published in Genomics. 2004 Apr;83(4):706-16.


Functional implications of calcium permeability of the channel formed by pannexin 1.

Vanden Abeele F, Bidaux G, Gordienko D, Beck B, Panchin YV, Baranova AV, Ivanov DV, Skryma R, Prevarskaya N.

Although human pannexins (PanX) are homologous to gap junction molecules, their physiological function in vertebrates remains poorly understood. Our results demonstrate that overexpression of PanX1 results in the formation of Ca(2+)-permeable gap junction channels between adjacent cells, thus, allowing direct intercellular Ca(2+) diffusion and facilitating intercellular Ca(2+) wave propagation. More intriguingly, our results strongly suggest that PanX1 may also form Ca(2+)-permeable channels in the endoplasmic reticulum (ER). These channels contribute to the ER Ca(2+) leak and thereby affect the ER Ca(2+) load. Because leakage remains the most enigmatic of those processes involved in intracellular calcium homeostasis, and the molecular nature of the leak channels is as yet unknown, the results of this work provide new insight into calcium signaling mechanisms. These results imply that for vertebrates pannexins may not simply duplicate the connexin function but may also provide additional pathways for intra- and intercellular calcium signaling and homeostasis.

This study has been published in J Cell Biol. 2006 Aug 14;174(4):535-46.


What is hidden in the pannexin treasure trove: the sneak peek and the guesswork.

Litvin O, Tiunova A, Connell-Alberts Y, Panchin Y, Baranova A.

Review paper summarizing our understanding of pannexins.

Connexins had been considered to be the only class of the vertebrate proteins capable of gap junction formation; however, new candidates for this function with no homology to connexins, termed pannexins were discovered. So far three pannexins were described in rodent and human genomes: Panx1, Panx2 and Panx3. Expressions of pannexins can be detected in numerous brain structures, and now found both in neuronal and glial cells. Hypothetical roles of pannexins in the nervous system include participating in sensory processing, hippocampal plasticity, synchronization between hippocampus and cortex, and propagation of the calcium waves supported by glial cells, which help maintain and modulate neuronal metabolism. Pannexin also may participate in pathological reactions of the neural cells, including their damage after ischemia and subsequent cell death. Recent study revealed non-gap junction function of Panx1 hemichannels in erythrocytes, where they serve as the conduits for the ATP release in response to the osmotic stress. High-throughput studies produced some evidences of the pannexin involvement in the process of tumorigenesis. According to brain cancer gene expression database REMBRANDT, PANX2 expression levels can predict post diagnosis survival for patients with glial tumors. Further investigations are needed to verify or reject hypotheses listed.

This review has been published in J Cell Mol Med. 2006 Jul-Sep;10(3):613-34.

A Role of Pannexins in the Development of the Astrocytic Tumors

PhD Student: Yvette Connell-Alberts

This is a collaborative project with the lab of Dr. K. Reilly (NCI Frederick)

It is well known that GJs are involved in tissue homeostasis and that they regulate and control cell proliferation, differentiation, and apoptosis, although the mechanistic aspects of these actions remain largely unknown. Disruption or alteration of such communications may lead to aberrant cell growth and tumor development. Connexin-encoding genes could serve as tumor suppressors. For example, transformed cells transduced with connexin genes have been shown to regain proliferation control, thereby suggesting that connexins do in fact have tumor-suppressor activity.

The most conclusive evidence of connexin depletion in tumorigenesis was recently collected for gliomas and glioblastomas. However, GJs have also been shown to stimulate the invasion of malignant gliomas. This picture became even more complex after the discovery of pannexins. Pannexins exhibit a remarkable sensitivity to blockade by CBX, a classical inhibitor of GJ communication. It is quite possible that CBX-inhibited cell migration of glioma cells described before is due to a suppression of pannexin channels

Evidence of pannexin involvement in the process of tumorigenesis is provided by high-throughput studies revealing differential expression of pannexin encoding genes in tumor samples and in model systems. A search for pannexin encoding genes in the brain cancer gene expression database REMBRANDT (Repository of Molecular Brain Neoplasia Data, http://rembrandt.nci.nih.gov/rembrandt) yields statistically significant PANX2-based predictions of post diagnosis survival for patients with glial tumors (Fig. 1). These findings are further strengthened by the observation that the human PANX2 gene is located within chromosomal region 22q13.3 which is often deleted in human astrocytomas and ependymomas. Thus, PANX2 could be viewed as a candidate tumor suppressor gene involved in gliomagenesis. On the top of that, in one recent study a Panx2 proteolitic fragment present into the bloodstream was identified as a potential biomarker representing a part of the serum signature of ovarian carcinoma.

Despite the obvious suggestion that the release of Panx2 fragments into the bloodstream of ovarian cancer patients may simply accompany the paraneoplastic process in some non-malignant tissues, it is tempting to speculate that an underlying loss of GJs is relevant to at least some manifestations of this malignancy. Evidences of PANX1 and PANX2 involvement in tumorigenesis are even less convincing. Nevertheless, we will review them here, as they still might point us to an interesting facet of pannexin polyfunctionality. High-throughput microarray analysis of the mouse hepatocarcinoma cell line Hca-F with a metastasis rate over 70% and its syngeneic cell line Hca-P with a metastasis rate less than 30% in order, revealed that Panx1 over-expression is characteristic for metastatic cancer spread. PANX1 gene amplification leading to its overexpression has been found in 2 out of 5 multiple myeloma cell lines studied, corroborating the hypothesis that this gene can serve as an oncogene. However, some other studies contradict to this point of view. The amount of PANX1 mRNA greatly increases after the inhibition of the low-grade serous carcinoma MPSC1 cell proliferation as measured by LongSAGE procedure. The MPSC1 cell line possesses a BRAF mutation that leads to constitutive activation (phosphorylation) of its downstream target, mitogen-activated protein kinase (MAPK), also known as extracellular signal regulated protein kinase (ERK). This activation could be prevented by treatment with a highly potent and selective inhibitor of MEK1/2, CI- 1040 (PD184352) leading to the profound suppression of the proliferation. Pronounced over-expression of Panx1 after CI-1040 treatment was confirmed by Real-Time PCR both in MPSC1 cell line and in two other ovarian carcinoma cell lines that contain activating KRAS mutations.

The only observation linking pannexin 3 to the tumorigenesis is an identification of this protein as a molecular partner of BCL6, a transcription factor involved in lymphomagenesis. This finding came from co-immunoprecipitation experiments with an anti-BCL6 antibody and subsequent tandem MS/MS spectroscopy.

At this point, it is difficult to say whether pannexins can be viewed as tumor suppressors or oncogenes unequivocally. Most likely, pannexins represent yet another group of the genes whose expression is disturbed in tumors. Similarly to connexins, particular pannexins may restrain initial growth of the tumor in situ, but hasten the metastatic spread of the existing malignancy. Future studies of the pannexin connection to cancer are warranted.

    Figure 1. Expression of the PANX2 mRNA in a human brain tumor samples. A. Gene expression plot that displays average expression intensities for the for PANX2 gene based on Affymetrix GeneChip arrays (U133 Plus 2.0 arrays) expression in human brain tumor samples. B. Kaplan-Meier Survival Plot for Samples with Differential PANX2 Gene Expression measured by unified probeset reporter 56666. Down-Regulated vs. Intermediate: P < 0.023; Down- Regulated vs. all other samples, P < 0.0194; Intermediate vs. all other samples, P < 0.0325. The log rank p-values are calculated using Mantel-Haenszel procedure.