Endogenous oxidized DNA bases and APE1 regulate the formation of G-quadruplex structures in the genome.

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.

Inhibition of Pyk2 and Src activity improves Cx43 gap junction intercellular communication.

Identification of proteins that interact with Cx43 has been instrumental in the understanding of gap junction (GJ) regulation. An in vitro phosphorylation screen identified that Protein tyrosine kinase 2 beta (Pyk2) phosphorylated purified Cx43CT and this led us to characterize the impact of this phosphorylation on Cx43 function. Mass spectrometry identified Pyk2 phosphorylates Cx43 residues Y247, Y265, Y267, and Y313. Western blot and immunofluorescence staining using HeLaCx43 cells, HEK 293 T cells, and neonatal rat ventricular myocytes (NRVMs) revealed Pyk2 can be activated by Src and active Pyk2 interacts with Cx43 at the plasma membrane. Overexpression of Pyk2 increases Cx43 phosphorylation and knock-down of Pyk2 decreases Cx43 phosphorylation, without affecting the level of active Src. In HeLaCx43 cells treated with PMA to activate Pyk2, a decrease in Cx43 GJ intercellular communication (GJIC) was observed when assayed by dye transfer. Moreover, PMA activation of Pyk2 could be inhibited by the small molecule PF4618433. This partially restored GJIC, and when paired with a Src inhibitor, returned GJIC to the no PMA control-level. The ability of Pyk2 and Src inhibitors to restore Cx43 function in the presence of PMA was also observed in NRVMs. Additionally, an animal model of myocardial infarction induced heart failure showed a higher level of active Pyk2 activity and increased interaction with Cx43 in ventricular myocytes. Src inhibitors have been used to reverse Cx43 remodeling and improve heart function after myocardial infarction; however, they alone could not fully restore proper Cx43 function. Our data suggest that Pyk2 may need to be inhibited, in addition to Src, to further (if not completely) reverse Cx43 remodeling and improve intercellular communication.

Regulation of Connexin32 by ephrin receptors and T-cell protein-tyrosine phosphatase.

Gap junctions are intercellular conduits that permit the passage of ions, small metabolites, and signaling molecules between cells. Connexin32 (Cx32) is a major gap junction protein in the liver and brain. Phosphorylation is integral to regulating connexin assembly, degradation, and electrical and metabolic coupling, as well as to interactions with molecular partners. Cx32 contains two intracellular tyrosine residues, and tyrosine phosphorylation of Cx32 has been detected after activation of the epidermal growth factor receptor; however, the specific tyrosine residue and the functional implication of this phosphorylation remain unknown. To address the limited available information on Cx32 regulation by tyrosine kinases, here we used the Cx32 C-terminal (CT) domain in an in vitro kinase-screening assay, which identified ephrin (Eph) receptor family members as tyrosine kinases that phosphorylate Cx32. We found that EphB1 and EphA1 phosphorylate the Cx32CT domain residue Tyr243 Unlike for Cx43, the tyrosine phosphorylation of the Cx32CT increased gap junction intercellular communication. We also demonstrated that T-cell protein-tyrosine phosphatase dephosphorylates pTyr243 The data presented above along with additional examples throughout the literature of gap junction regulation by kinases, indicate that one cannot extrapolate the effect of a kinase on one connexin to another.

Phosphorylation of Cx43 residue Y313 by Src contributes to blocking the interaction with Drebrin and disassembling gap junctions.

Phosphorylation regulates connexin43 (Cx43) function from assembly/disassembly to coupling at the plaque. Src is a tyrosine kinase known to both phosphorylate Cx43 (residues Y247 and Y265) and affect gap junction intercellular communication. However, the Cx43 carboxyl-terminal (CT) domain contains additional tyrosine residues and proteomic discovery mass spectrometry data identified Y313 as a potential phosphorylation target. Based upon the study of Lin et al. (2001) J. Cell Biol., which still observed tyrosine phosphorylation by Src when using a Cx43 Y247/Y265F mutant, we addressed the possibility of Y313 phosphorylation (pY313) by Src. In vitro Src phosphorylation of purified Cx43CT followed by mass spectroscopy revealed that Src also phosphorylates Y313. This observation was confirmed by repeating the in vitro phosphorylation using different combinations of Cx43CT Y → F mutants and a general anti-pTyr antibody. Next, a phospho-specific antibody was generated to help characterize the importance of pY313. We established an in cyto experimental system by stably expressing Cx43 WT and mutants (Y247F, Y265F, Y313F, Y247/265F, Y247/313F, Y265/313F, or Y247/265/313F) in Cx43-deficient HeLa cells. Cx43 WT and mutants, in the absence of v-Src, localized to the plasma membrane and formed gap junctions. When v-Src was over-expressed, Cx43 WT localized intracellularly, while all of the single and double mutants remained able to form plaques and transfer dye, albeit variable in number and amount, respectively. Complete Src-resistance was only achieved with the Cx43 Y247/265/313F mutant. Furthermore, Cx43 Y265F inhibited the ability of v-Src to phosphorylate Y247 and Y313 as well as phosphorylation at both Y265 and Y313 was necessary to inhibit the Cx43 interaction with Drebrin. Finally, we observed in diseased cardiac tissue, in which Src is active, an increase in intercalated disc and intracellular localized Cx43 pY313.

Regulation of Cx37 channel and growth-suppressive properties by phosphorylation.

Growth suppression mediated by connexin 37 (Cx37; also known as GJA4) requires interaction between its C-terminus and functional pore-forming domain. Using rat insulinoma cells, we show that Cx37 induces cell death and cell cycle arrest, and slowed cell cycling. Whether differential phosphorylation might regulate intramolecular interactions, and consequently the growth-suppressive phenotype, is unknown. Protein kinase C inhibition increased the open state probability of low-conductance gap junction channels (GJChs) and reduced GJCh closed state probability. Substituting alanine at serine residues 275, 302 and 328 eliminated Cx37-induced cell death, supported proliferation and reduced the GJCh closed state probability. With additional alanine for serine substitutions at residues 285, 319, 321 and 325, Cx37-induced cell death was eliminated and the growth arrest period prolonged, and GJCh closed state probability was restored. With aspartate substitution at these seven sites, apoptosis was induced and the open state probability of large conductance GJChs (and hemichannels) was increased. These data suggest that differential phosphorylation of the C-terminus regulates channel conformation and, thereby, cell cycle progression and cell survival.

Protein⁻Protein Interactions with Connexin 43: Regulation and Function.

Connexins are integral membrane building blocks that form gap junctions, enabling direct cytoplasmic exchange of ions and low-molecular-mass metabolites between adjacent cells. In the heart, gap junctions mediate the propagation of cardiac action potentials and the maintenance of a regular beating rhythm. A number of connexin interacting proteins have been described and are known gap junction regulators either through direct effects (e.g., kinases) or the formation of larger multifunctional complexes (e.g., cytoskeleton scaffold proteins). Most connexin partners can be categorized as either proteins promoting coupling by stimulating forward trafficking and channel opening or inhibiting coupling by inducing channel closure, internalization, and degradation. While some interactions have only been implied through co-localization using immunohistochemistry, others have been confirmed by biophysical methods that allow detection of a direct interaction. Our understanding of these interactions is, by far, most well developed for connexin 43 (Cx43) and the scope of this review is to summarize our current knowledge of their functional and regulatory roles. The significance of these interactions is further exemplified by demonstrating their importance at the intercalated disc, a major hub for Cx43 regulation and Cx43 mediated effects.

Connexin43 Carboxyl-Terminal Domain Directly Interacts with ß-Catenin.

Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of ß-catenin, and results in the enhanced accumulation of the Cx43 protein and the formation of gap junction channels. In response to Wnt signaling, ß-catenin co-localizes with the Cx43 protein itself as part of a complex at the gap junction plaque. Work from several labs have also shown indirect evidence of this interaction via reciprocal co-immunoprecipitation. Our goal for the current study was to identify whether ß-catenin directly interacts with Cx43, and if so, the location of that direct interaction. Identifying residues involved in direct protein⁻protein interaction is of importance when they are correlated to the phosphorylation of Cx43, as phosphorylation can modify the binding affinities of Cx43 regulatory protein partners. Therefore, combining the location of a protein partner interaction on Cx43 along with the phosphorylation pattern under different homeostatic and pathological conditions will be crucial information for any potential therapeutic intervention. Here, we identified that ß-catenin directly interacts with the Cx43 carboxyl-terminal domain, and that this interaction would be inhibited by the Src phosphorylation of Cx43CT residues Y265 and Y313.

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization.

Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes. To gain insight regarding these determinants, we focused on the carboxyl-terminal (CT) domain because of its importance in channel regulation and large degree of sequence divergence among connexin family members. Using in vitro biophysical experiments, we identified a structural feature unique to Cx45: high affinity (KD~100nM) dimerization between CT domains. In this study, we sought to determine if this dimerization occurs in cells and to identify the biological significance of the dimerization. Using a bimolecular fluorescence complementation assay, we demonstrate that the CT domains dimerize at the plasma membrane. By inhibiting CT dimerization with a mutant construct, we show that CT dimerization is necessary for proper Cx45 membrane localization, turnover, phosphorylation status, and binding to protein partners. Furthermore, CT dimerization is needed for normal intercellular communication and hemichannel activity. Altogether, our results demonstrate that CT dimerization is a structural feature important for correct Cx45 function. This study is significant because discovery of how interactions mediated by the CT domains can be modulated would open the door to strategies to ameliorate the pathological effects of altered connexin regulation in the failing heart.

Regulation of Connexin43 Function and Expression by Tyrosine Kinase 2.

Connexin43 (Cx43) assembly and degradation, the regulation of electrical and metabolic coupling, as well as modulating the interaction with other proteins, involve phosphorylation. Here, we identified and characterized the biological significance of a novel tyrosine kinase that phosphorylates Cx43, tyrosine kinase 2 (Tyk2). Activation of Tyk2 led to a decrease in Cx43 gap junction communication by increasing the turnover rate of Cx43 from the plasma membrane. Tyk2 directly phosphorylated Cx43 residues Tyr-247 and Tyr-265, leading to indirect phosphorylation on residues Ser-279/Ser-282 (MAPK) and Ser-368 (PKC). Although this phosphorylation pattern is similar to what has been observed following Src activation, the response caused by Tyk2 occurred when Src was inactive in NRK cells. Knockdown of Tyk2 at the permissive temperature (active v-Src) in LA-25 cells decreased Cx43 phosphorylation, indicating that although activation of Tyk2 and v-Src leads to phosphorylation of the same Cx43CT residues, they are not identical in level at each site. Additionally, angiotensin II activation of Tyk2 increased the intracellular protein level of Cx43 via STAT3. These findings indicate that, like Src, Tyk2 can also inhibit gap junction communication by phosphorylating Cx43.

Structural Studies of the Nedd4 WW Domains and Their Selectivity for the Connexin43 (Cx43) Carboxyl Terminus.

Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) was the first ubiquitin protein ligase identified to interact with connexin43 (Cx43), and its suppressed expression results in accumulation of gap junction plaques at the plasma membrane. Nedd4-mediated ubiquitination of Cx43 is required to recruit Eps15 and target Cx43 to the endocytic pathway. Although the Cx43 residues that undergo ubiquitination are still unknown, in this study we address other unresolved questions pertaining to the molecular mechanisms mediating the direct interaction between Nedd4 (WW1-3 domains) and Cx43 (carboxyl terminus (CT)). All three WW domains display a similar three antiparallel ß-strand structure and interact with the same Cx43CT(283)PPXY(286)sequence. Although Tyr(286)is essential for the interaction, MAPK phosphorylation of the preceding serine residues (Ser(P)(279)and Ser(P)(282)) increases the binding affinity by 2-fold for the WW domains (WW2 > WW3 ≫ WW1). The structure of the WW2·Cx43CT(276-289)(Ser(P)(279), Ser(P)(282)) complex reveals that coordination of Ser(P)(282)with the end of ß-strand 3 enables Ser(P)(279)to interact with the back face of ß-strand 3 (Tyr(286)is on the front face) and loop 2, forming a horseshoe-shaped arrangement. The close sequence identity of WW2 with WW1 and WW3 residues that interact with the Cx43CT PPXY motif and Ser(P)(279)/Ser(P)(282)strongly suggests that the significantly lower binding affinity of WW1 is the result of a more rigid structure. This study presents the first structure illustrating how phosphorylation of the Cx43CT domain helps mediate the interaction with a molecular partner involved in gap junction regulation.

TC-PTP directly interacts with connexin43 to regulate gap junction intercellular communication.

Protein kinases have long been reported to regulate connexins; however, little is known about the involvement of phosphatases in the modulation of intercellular communication through gap junctions and the subsequent downstream effects on cellular processes. Here, we identify an interaction between the T-cell protein tyrosine phosphatase (TC-PTP, officially known as PTPN2) and the carboxyl terminus of connexin43 (Cx43, officially known as GJA1). Two cell lines, normal rat kidney (NRK) cells endogenously expressing Cx43 and an NRK-derived cell line expressing v-Src with temperature-sensitive activity, were used to demonstrate that EGF and v-Src stimulation, respectively, induced TC-PTP to colocalize with Cx43 at the plasma membrane. Cell biology experiments using phospho-specific antibodies and biophysical assays demonstrated that the interaction is direct and that TC-PTP dephosphorylates Cx43 residues Y247 and Y265, but does not affect v-Src. Transfection of TC-PTP also indirectly led to the dephosphorylation of Cx43 S368, by inactivating PKCα and PKCδ, with no effect on the phosphorylation of S279 and S282 (MAPK-dependent phosphorylation sites). Dephosphorylation maintained Cx43 gap junctions at the plaque and partially reversed the channel closure caused by v-Src-mediated phosphorylation of Cx43. Understanding dephosphorylation, along with the well-documented roles of Cx43 phosphorylation, might eventually lead to methods to modulate the regulation of gap junction channels, with potential benefits for human health.

Secondary structural analysis of the carboxyl-terminal domain from different connexin isoforms.

The connexin carboxyl-terminal (CxCT) domain plays a role in the trafficking, localization, and turnover of gap junction channels, as well as the level of gap junction intercellular communication via numerous post-translational modifications and protein-protein interactions. As a key player in the regulation of gap junctions, the CT presents itself as a target for manipulation intended to modify function. Specific to intrinsically disordered proteins, identifying residues whose secondary structure can be manipulated will be critical toward unlocking the therapeutic potential of the CxCT domain. To accomplish this goal, we used biophysical methods to characterize CxCT domains attached to their fourth transmembrane domain (TM4). Circular dichroism and nuclear magnetic resonance were complementary in demonstrating the connexin isoforms that form the greatest amount of α-helical structure in their CT domain (Cx45 > Cx43 > Cx32 > Cx50 > Cx37 ≈ Cx40 ≈ Cx26). Studies compared the influence of 2,2,2-trifluoroethanol, pH, phosphorylation, and mutations (Cx32, X-linked Charcot-Marie Tooth disease; Cx26, hearing loss) on the TM4-CxCT structure. While pH modestly influences the CT structure, a major structural change was associated with phosphomimetic substitutions. Since most connexin CT domains are phosphorylated throughout their life cycle, studies of phospho-TM4-CxCT isoforms will be critical toward understanding the role that structure plays in regulating gap junction function.

Degradation of gap junction connexins is regulated by the interaction with Cx43-interacting protein of 75 kDa (CIP75).

Connexins are a family of transmembrane proteins that form gap junction channels. These proteins undergo both proteasomal and lysosomal degradation, mechanisms that serve to regulate connexin levels. Our previous work described CIP75 [connexin43 (Cx43)-interacting protein of 75 kDa], a protein involved in proteasomal degradation, as a novel Cx43-interacting protein. We have discovered two additional connexins, connexin40 (Cx40) and connexin45 (Cx45), that interact with CIP75. Nuclear magnetic resonance (NMR) analyses identified the direct interaction of the CIP75 UBA domain with the carboxyl-terminal (CT) domains of Cx40 and Cx45. Reduction in CIP75 by shRNA in HeLa cells expressing Cx40 or Cx45 resulted in increased levels of the connexins. Furthermore, treatment with trafficking inhibitors confirmed that both connexins undergo endoplasmic reticulum-associated degradation (ERAD), and that CIP75 preferentially interacts with the connexin proteins bound for proteasomal degradation from the ER. In addition, we have also discovered that CIP75 interacts with ER-localized Cx32 in a process that is likely mediated by Cx32 ubiquitination. Thus, we have identified novel interacting connexin proteins of CIP75, indicating a role for CIP75 in regulating the levels of connexins in general, through proteasomal degradation.

Characterization of the connexin45 carboxyl-terminal domain structure and interactions with molecular partners.

Mechanisms underlying the initiation and persistence of lethal cardiac rhythms are of significant clinical and scientific interests. Gap junctions are principally involved in forming the electrical connections between myocytes, and changes in distribution, density, and properties are consistent characteristics in arrhythmic heart disease. Therefore, understanding the structure and function of gap junctions during normal and abnormal impulse propagation are essential in the control of arrhythmias. For example, Cx45 is predominately expressed in the specialized myocytes of the impulse generation and conduction system. In both ventricular and atrial human working myocytes, Cx45 is present in very low quantities. However, a reduction in Cx43 coupled with an increased Cx45 protein levels within the ventricles have been observed after myocardial infarction and end-stage heart failure. Cx45 may influence electrical and/or metabolic coupling as a result of pathophysiological overexpression. Our goal was to identify mechanisms that could cause cellular coupling to be different between the cardiac connexins. Based upon the conserved transmembrane and extracellular loop segments, our focus was on identifying features within the divergent cytoplasmic portions. Here, we biophysically characterize the carboxyl-terminal domain of Cx45 (Cx45CT). Purification revealed the possibility of oligomeric species, which was confirmed by analytical ultracentrifugation experiments. Sedimentation equilibrium and circular dichroism studies of different Cx45CT constructs identified one region of α-helical structure (A333-N361) that mediates CT dimerization through hydrophobic contacts. Interestingly, the binding affinity of Cx45CT dimerization is 1000-fold stronger than Cx43CT dimerization. Cx45CT resonance assignments were also used to identify the binding sites and affinities of molecular partners involved in the Cx45 regulation; although none disrupted dimerization, many of these proteins interacted within one intrinsically disordered region (P278-P285). This domain has similarities with other cardiac connexins, and we propose they constitute a master regulatory domain, which contains overlapping molecular partner binding, cis-trans proline isomerization, and phosphorylation sites.

Rabankyrin-5 interacts with EHD1 and Vps26 to regulate endocytic trafficking and retromer function.

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.

Differential roles of C-terminal Eps15 homology domain proteins as vesiculators and tubulators of recycling endosomes.

Endocytic recycling involves the return of membranes and receptors to the plasma membrane following their internalization into the cell. Recycling generally occurs from a series of vesicular and tubular membranes localized to the perinuclear region, collectively known as the endocytic recycling compartment. Within this compartment, receptors are sorted into tubular extensions that later undergo vesiculation, allowing transport vesicles to move along microtubules and return to the cell surface where they ultimately undergo fusion with the plasma membrane. Recent studies have led to the hypothesis that the C-terminal Eps15 homology domain (EHD) ATPase proteins are involved in the vesiculation process. Here, we address the functional roles of the four EHD proteins. We developed a novel semipermeabilized cell system in which addition of purified EHD proteins to reconstitute vesiculation allows us to assess the ability of each protein to vesiculate MICAL-L1-decorated tubular recycling endosomes (TREs). Using this assay, we show that EHD1 vesiculates membranes, consistent with enhanced TRE generation observed upon EHD1 depletion. EHD4 serves a role similar to that of EHD1 in TRE vesiculation, whereas EHD2, despite being capable of vesiculating TREs in the semipermeabilized cells, fails to do so in vivo. Surprisingly, the addition of EHD3 causes tubulation of endocytic membranes in our semipermeabilized cell system, consistent with the lack of tubulation observed upon EHD3 depletion. Our novel vesiculation assay and in vitro electron microscopy analysis, combined with in vivo data, provide evidence that the functions of both EHD1 and EHD4 are primarily in TRE membrane vesiculation, whereas EHD3 is a membrane-tubulating protein.

Effects of phosphorylation on the structure and backbone dynamics of the intrinsically disordered connexin43 C-terminal domain.

Phosphorylation of the connexin43 C-terminal (Cx43CT) domain regulates gap junction intercellular communication. However, an understanding of the mechanisms by which phosphorylation exerts its effects is lacking. Here, we test the hypothesis that phosphorylation regulates Cx43 gap junction intercellular communication by mediating structural changes in the C-terminal domain. Circular dichroism and nuclear magnetic resonance were used to characterize the effects of phosphorylation on the secondary structure and backbone dynamics of soluble and membrane-tethered Cx43CT domains. Cx43CT phospho-mimetic isoforms, which have Asp substitutions at specific Ser/Tyr sites, revealed phosphorylation alters the α-helical content of the Cx43CT domain only when attached to the membrane. The changes in secondary structure are due to variations in the conformational preference and backbone flexibility of residues adjacent and distal to the site(s) of modification. In addition to the known direct effects of phosphorylation on molecular partner interactions, the data presented here suggest phosphorylation may also indirectly regulate binding affinity by altering the conformational preference of the Cx43CT domain.

Evaluation of Tyrosine Kinase Inhibitors Loaded Injectable Hydrogels for Improving Connexin43 Gap Junction Intercellular Communication.

Myocardial infarction (MI) is one of the leading causes of death in the developed world, and the loss of cardiomyocytes plays a critical role in the pathogenesis of heart failure. Implicated in this process is a decrease in gap junction intercellular communication due to remodeling of Connexin43 (Cx43). We previously identified that intraperitoneal injection of the Pyk2 inhibitor PF4618433 reduced infarct size, maintained Cx43 at the intercalated disc in left ventricle hypertrophic myocytes, and improved cardiac function in an MI animal model of heart failure. With the emergence of injectable hydrogels as a therapeutic toward the regeneration of cardiac tissue after MI, here, we provide proof of concept that the release of tyrosine kinase inhibitors from hydrogels could have beneficial effects on cardiomyocytes. We developed an injectable hydrogel consisting of thiolated hyaluronic acid and P123-maleimide micelles that can incorporate PF4618433 as well as the Src inhibitor Saracatinib and achieved sustained release (of note, Src activates Pyk2). Using neonatal rat ventricular myocytes in the presence of a phorbol ester, endothelin-1, or phenylephrine to stimulate cardiac hypertrophy, the release of PF4618433 from the hydrogel had the same ability to decrease Cx43 tyrosine phosphorylation and maintain Cx43 localization at the plasma membrane as when directly added to the growth media. Additional beneficial effects included decreases in apoptosis, the hypertrophic marker atrial natriuretic peptide (ANP), and serine kinases upregulated in hypertrophy. Finally, the presence of both PF4618433 and Saracatinib further decreased the level of ANP and apoptosis than each inhibitor alone, suggesting that a combinatorial approach may be most beneficial. These findings provide the groundwork to test if tyrosine kinase inhibitor release from hydrogels will have a beneficial effect in an animal model of MI-induced heart failure.

Carboxy terminus and pore-forming domain properties specific to Cx37 are necessary for Cx37-mediated suppression of insulinoma cell proliferation.

Connexin 37 (Cx37) suppresses cell proliferation when expressed in rat insulinoma (Rin) cells, an effect also manifest in vivo during vascular development and in response to tissue injury. Mutant forms of Cx37 with nonfunctional channels but normally localized, wild-type carboxy termini are not growth suppressive. Here we determined whether the carboxy-terminal (CT) domain is required for Cx37-mediated growth suppression and whether the Cx37 pore-forming domain can be replaced with the Cx43 pore-forming domain and still retain growth-suppressive properties. We show that despite forming functional gap junction channels and hemichannels, Cx37 with residues subsequent to 273 replaced with a V5-epitope tag (Cx37-273tr*V5) had no effect on the proliferation of Rin cells, did not facilitate G1-cell cycle arrest with serum deprivation, and did not prolong cell cycle time comparably to the wild-type protein. The chimera Cx43*CT37, comprising the pore-forming domain of Cx43 and CT of Cx37, also did not suppress proliferation, despite forming functional gap junctions with a permselective profile similar to wild-type Cx37. Differences in channel behavior of both Cx37-273tr*V5 and Cx43*CT37 relative to their wild-type counterparts and failure of the Cx37-CT to interact as the Cx43-CT does with the Cx43 cytoplasmic loop suggest that the Cx37-CT and pore-forming domains are both essential to growth suppression by Cx37.

Regulation of Cx43 Gap Junction Intercellular Communication by Bruton's Tyrosine Kinase and Interleukin-2-Inducible T-Cell Kinase.

T and B cell receptor signaling involves the activation of Akt, MAPKs, and PKC as well as an increase in intracellular Ca2+ and calmodulin activation. While these coordinate the rapid turnover of gap junctions, also implicated in this process is Src, which is not activated as part of T and B cell receptor signaling. An in vitro kinase screen identified that Bruton's tyrosine kinase (BTK) and interleukin-2-inducible T-cell kinase (ITK) phosphorylate Cx43. Mass spectroscopy revealed that BTK and ITK phosphorylate Cx43 residues Y247, Y265, and Y313, which are identical to the residues phosphorylated by Src. Overexpression of BTK or ITK in the HEK-293T cells led to increased Cx43 tyrosine phosphorylation as well as decreased gap junction intercellular communication (GJIC) and Cx43 membrane localization. In the lymphocytes, activation of the B cell receptor (Daudi cells) or T cell receptor (Jurkat cells) increased the BTK and ITK activity, respectively. While this led to increased tyrosine phosphorylation of Cx43 and decreased GJIC, the cellular localization of Cx43 changed little. We have previously identified that Pyk2 and Tyk2 also phosphorylate Cx43 at residues Y247, Y265, and Y313 with a similar cellular fate to that of Src. With phosphorylation critical to Cx43 assembly and turnover, and kinase expression varying between different cell types, there would be a need for different kinases to achieve the same regulation of Cx43. The work presented herein suggests that in the immune system, ITK and BTK have the capacity for the tyrosine phosphorylation of Cx43 to alter the gap junction function in a similar manner as Pyk2, Tyk2, and Src.