Inflammation accompanies heart failure and is a mediator of cardiac fibrosis. CaMKIIδ plays an essential role in adverse remodeling and decompensation to heart failure. We postulated that inflammation is the mechanism by which CaMKIIδ contributes to adverse remodeling in response to nonischemic interventions. We demonstrate that deletion of CaMKIIδ in the cardiomyocyte (CKO) significantly attenuates activation of NF-κB, expression of inflammatory chemokines and cytokines, and macrophage accumulation induced by angiotensin II (Ang II) infusion. The inflammasome was activated by Ang II, and this response was also diminished in CKO mice. These events occurred prior to any evidence of Ang II-induced cell death. In addition, CaMKII-dependent inflammatory gene expression and inflammasome priming were observed as early as the third hour of infusion, a time point at which macrophage recruitment was not evident. Inhibition of either the inflammasome or monocyte chemoattractant protein 1 (MCP1) signaling attenuated macrophage accumulation, and these interventions, like cardiomyocyte CaMKIIδ deletion, diminished the fibrotic response to Ang II. Thus, activation of CaMKIIδ in the cardiomyocyte represents what we believe to be a novel mechanism for initiating inflammasome activation and an inflammatory gene program that leads to macrophage recruitment and ultimately to development of fibrosis.

Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are slowly disrupted and dismantled. 5-Fluorouracil (5-FU) and the camptothecin derivatives irinotecan and topotecan, are linked to altered nuclear transport of specific proteins; however, their general effects on the NPC and transport during apoptosis have not been characterized. We demonstrate that 5-FU, but not topotecan, increases NPC permeability, and disrupts Ran-mediated nuclear transport before the disruption of the NPC. This increased permeability is dependent on increased cellular calcium, as the Ca chelator BAPTA-AM, abolishes the effect. Furthermore, increased calcium alone was sufficient to disrupt the Ran gradient. Combination treatments of 5-FU with topotecan or irinotecan, similarly disrupted nuclear transport before disassembly of the NPC. In both single and combination treatments nuclear transport was disrupted before caspase 9 activation, indicating that 5-FU induces an early caspase-independent increase in NPC permeability and alteration of nuclear transport. Because Crm1-mediated nuclear export of tumor suppressors is linked to drug resistance we also examined the effect of 5-FU on the nuclear export of a specific target, topoisomerase. 5-FU treatment led to accumulation of topoisomerase in the nucleus and recovered the loss nuclear topoisomerase induced by irinotecan or topotecan, a known cause of drug resistance. Furthermore, 5-FU retains its ability to cause nuclear accumulation of p53 in the presence of irinotecan or topotecan. Our results reveal a new mechanism of action for these therapeutics during apoptosis, opening the door to other potential combination chemotherapies that employ 5-FU as a calcium mediated inhibitor of Crm1-induced nuclear export of tumor suppressors.

Regulation of nuclear transport is an essential component of apoptosis. As chemotherapy induced cell death progresses, nuclear transport and the nuclear pore complex (NPC) are disrupted and dismantled. Specifically, 5‐flurouracil (5‐FU) increases NPC permeability and disrupts the Ran gradient, decreasing it's nuclear levels and inhibiting nuclear export. As a result, combination of 5‐FU with other chemotherapeutics, such as topotican, increases their efficiency, by retaining pro‐apoptotic factors in the nucleus. Here we examine the ability of 5‐FU to enhance the effects of another drug, gemcitabine, previously shown to work synergistically with nuclear transport inhibitors. Interestingly, we found that while both 5‐FU and gemcitabine cause a decrease in nuclear Ran, combination of the drugs led to no significant change in Ran localization. In order to determine the mechanism of action for both drugs and the means of their counteraction, we explored their effects on NPC permeability. As expected, 5‐FU increased NPC permeability; however, gemcitabine did not disrupt the NPC. Additionally, gemcitabine counteracted 5‐FU, indicating that their antagonistic effects on Ran localization occur at the level of NPC permeability. As 5‐FU's effects on the NPC are known to be calcium dependent, we also explored whether gemcitabine similarly counteracts thapsigargin, an inhibitor of ER calcium uptake and known disruptor of nuclear Ran localization. Interestingly, gemcitabine had no effect on thapsigargin's ability to decrease nuclear Ran levels. Furthermore, thapsigargin had no effect on NPC permeability. Instead, thapsigargin counteracted the ability of 5‐FU to decrease nuclear Ran levels, despite the fact that it was unable to change 5‐FU induced NPC permeability. These results suggest that gemcitabine and thapsigargin mediate similar negative effects on Ran and nuclear transport counteractive to 5‐FU, but thapsigargin does so via an unknown mechanism. Therefore, the effects of 5‐FU and thapsigargin may reflect various potential means of calcium mediated changes to nuclear transport in early apoptosis. Future work will involve exploring the direct impact of these treatments on calcium levels. Importantly, the impacts of all drug treatments on Ran in the nucleus were due to changes in cellular localization differences alone, as immunoblot analysis demonstrated no changes in Ran protein expression levels. The combinatory effects of these drugs on the NPC and nuclear transport were unexpected based on previous work, and indicate the wide variation in nuclear disruption in apoptosis. At this level it is unclear if the impacts of 5‐FU, thapsigargin, and gemcitabine on nuclear transport and the NPC alone will be sufficient to efficiently predict the efficacy of these combinations on apoptosis. The counteraction of gemcitabine or thapsigargin on 5‐FU at this level may or may not be a predictor of cell death, and thus our ongoing work involves studies of cell viability and the localization of apoptotic factors with these treatments. Understanding the molecular mechanisms of these drugs will help to inform the usefulness of their combination in cancer treatment, particularly in cases of drug resistance.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The GTPase, Ran, is a major regulator of nuclear transport and disruption of its established gradient disrupts nuclear export and leads to cell death through the accumulation of pro‐apoptotic factors. The chemotherapy 5‐Fluorouracil (5‐FU) increases nuclear pore permeability causing Ran to leak into the cytoplasm, disrupting the gradient and in turn nuclear export, leading to cell death of HeLa cervical cancer cells. Our previous work demonstrated that combining 5‐FU with topoisomerase inhibitors can have a greater total impact on disruption of the Ran gradient and accumulates proteins in the nucleus. Gemcitabine (GEM) is widely used when treating pancreatic cancer; yet a resistance is common. However, combination of GEM with nuclear export inhibitors can help overcome this problem. Therefore, we sought to determine if combination of 5‐FU and GEM could impact nuclear export to help overcome GEM resistance. Our initial studies found that 5‐FU+GEM further decreased nuclear Ran in HeLa cells and lead to increased cell death in combination over GEM alone. Therefore, we transitioned to exploring the effects of 5‐FU+GEM on disrupting the Ran gradient in PANC1 pancreatic cells. Both 5‐FU and GEM individually and in combination decreased the nuclear Ran levels by 15%, 17%, and 23% respectively. This disruption of nuclear export may help overcome GEM resistance in PANC1 cells. Specifically, our ongoing work is exploring the impact of 5FU+GEM on the nuclear retention of the tumor suppressors p27 and p21 as well as cell viability to identify a possible mechanism for the impact of the 5‐FU+GEM combination on apoptosis. Our work helps support a possible mechanism for overcoming GEM resistance in Panc1 cells and identifies a potential combination treatment with 5‐FU for use against pancreatic cancer.

Altered nuclear transport plays a role in the mislocalization of tumor suppressor proteins in certain cancers. In addition, the disruption of nuclear transport and the nuclear pore complex during apoptosis are important mechanisms of action in several different chemotherapies. We previously demonstrated that the chemotherapy 5‐Fluorouracil (5‐FU) alters nuclear transport by disrupting the Ran gradient and increasing nuclear pore permeability. This block in nuclear transport may be advantageous to overcome Crm1‐induced excessive nuclear export in some cancers or with certain cases of drug resistance. Other work has shown that an additional chemotherapy, gemcitabine, is more effective when in combination with Crm1 inhibition. Therefore, we were interested in determining if gemcitabine and 5‐FU work effectively alone and in combination to disrupt nuclear transport via mislocalization of the transport protein Ran. We utilized immunofluorescence to determine the localization of Ran, which is normally localized to the nucleus. We show here that while each of these drugs mislocalized Ran at both 6h and 24h time points, and that gemcitabine counteracts the effects of 5‐FU in combination treatments to induce no change in Ran localization. This may be due to gemcitabine inhibiting 5‐FU's calcium‐mediated mechanism for inducing nuclear pore permeability. Additionally, we were interested in determining how these drugs affected the localization of three cargo proteins regulated by Crm1‐mediated export: p53, p27 and p21. These proteins have important function in the nucleus to control cell cycle progression and induction of apoptosis. Therefore, inhibition of Crm1 export should lead to nuclear accumulation of these factors and a potential increase in their function as tumor suppressors. We show here that 5‐FU and gemcitabine have opposing effects on the nuclear localization of both p53 and p21, and that the drugs again have counteractive effects when in combination treatments. Both 5‐FU and gemcitabine decrease the nuclear localization and expression of p27, while the combination treatment shows an additive effect on both localization and expression. The effects of these drugs on protein localization may be related to an additional block in nuclear import, as well as an induction of nuclear pore permeability downstream of the induction of apoptosis. Further research will help to characterize the effects of 5‐FU and gemcitabine on nuclear transport and subsequent protein localization. This may help to determine how these drugs may be more effectively used in the treatment of specific cancers and in specific drug combinations.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.