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Heat Shock Inhibition of CDK5 Increases NOXA Levels through miR-23a Repression*

Open AccessPublished:March 31, 2015DOI:https://doi.org/10.1074/jbc.M114.625988
      Hyperthermia is a proteotoxic stress that is lethal when exposure is extreme but also cytoprotective in that sublethal exposure leads to the synthesis of heat shock proteins, including HSP70, which are able to inhibit stress-induced apoptosis. CDK5 is an atypical cyclin-dependent kinase family member that regulates many cellular functions including motility and survival. Here we show that exposure of a human lymphoid cell line to hyperthermia causes CDK5 insolubilization and loss of tyrosine-15 phosphorylation, both of which were prevented in cells overexpressing HSP70. Inhibition of CDK5 activity with roscovitine-sensitized cells to heat induced apoptosis indicating a protective role for CDK5 in inhibiting heat-induced apoptosis. Both roscovitine and heat shock treatment caused increased accumulation of NOXA a pro-apoptotic BH3-only member of the BCL2 family. The increased abundance of NOXA by CDK5 inhibition was not a result of changes in NOXA protein turnover. Instead, CDK5 inhibition increased NOXA mRNA and protein levels by decreasing the expression of miR-23a, whereas overexpressing the CDK5 activator p35 attenuated both of these effects on NOXA and miR-23a expression. Lastly, overexpression of miR-23a prevented apoptosis under conditions in which CDK5 activity was inhibited. These results demonstrate that CDK5 activity provides resistance to heat-induced apoptosis through the expression of miR-23a and subsequent suppression of NOXA synthesis. Additionally, they indicate that hyperthermia induces apoptosis through the insolubilization and inhibition of CDK5 activity.
      Background: HSP70 inhibits heat-induced apoptosis by preventing NOXA accumulation.
      Results: Inhibition of CDK5 in heat-shocked cells reduces levels of miR-23a leading to increased NOXA abundance, which is prevented by HSP70.
      Conclusion: CDK5 regulates miR-23a expression and affects NOXA abundance.
      Significance: The prosurvival function of HSP70 can be attributed to its ability to prevent the stress-induced inhibition of CDK5.

      Introduction

      Cyclin-dependent kinase 5 (CDK5)
      The abbreviations used are: CDK
      cyclin-dependent kinase
      BH3-only
      BCL2 family protein containing only the BCL2 homology domain number 3
      pY15-CDK5
      CDK5 phosphorylated on tyrosine residue number 15
      HSP
      heat shock protein
      RT-qPCR
      reverse transcription-quantitative real-time polymerase chain reaction
      pri/pre-miRNA
      primary/precursor microRNA.
      is a member of the cyclin-dependent kinase family with unique roles unrelated to cell cycle progression. It shares sequence homology with typical CDKs, such as CDK1, and phosphorylates substrates having the same consensus motif ((S/T)PX(K/H/R)), however its activity is dependent upon the binding of the activators p35 (CDK5R1) or p39 (CDK5R2), which are not cyclins but do contain a cyclin box necessary for CDK5 binding (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ,
      • Shah K.
      • Lahiri D.K.
      Cdk5 activity in the brain - multiple paths of regulation.
      ). CDK5 is also atypical in that threonine phosphorylation of the activation loop is not required for its activity and its activity is not inhibited by Tyr-15 phosphorylation. Instead, phosphorylation of Tyr-15 by the non-receptor tyrosine kinases c-Abl and Fyn increases CDK5 activity (
      • Sasaki Y.
      • Cheng C.
      • Uchida Y.
      • Nakajima O.
      • Ohshima T.
      • Yagi T.
      • Taniguchi M.
      • Nakayama T.
      • Kishida R.
      • Kudo Y.
      • Ohno S.
      • Nakamura F.
      • Goshima Y.
      Fyn and Cdk5 mediate semaphorin-3A signaling, which is involved in regulation of dendrite orientation in cerebral cortex.
      ,
      • Zukerberg L.R.
      • Patrick G.N.
      • Nikolic M.
      • Humbert S.
      • Wu C.L.
      • Lanier L.M.
      • Gertler F.B.
      • Vidal M.
      • Van Etten R.A.
      • Tsai L.H.
      Cables links Cdk5 and c-Abl and facilitates Cdk5 tyrosine phosphorylation, kinase upregulation, and neurite outgrowth.
      ). CDK5 was originally believed to be restricted to neuronal cells where it plays fundamental roles in brain development through regulation of cell migration, neurite outgrowth, and synapse formation (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ,
      • Shah K.
      • Lahiri D.K.
      Cdk5 activity in the brain - multiple paths of regulation.
      ). However, CDK5 also regulates numerous activities in a wide variety of non-neuronal cells including lymphocytes (
      • Arif A.
      Extraneuronal activities and regulatory mechanisms of the atypical cyclin-dependent kinase Cdk5.
      ,
      • Contreras-Vallejos E.
      • Utreras E.
      • Gonzalez-Billault C.
      Going out of the brain: non-nervous system physiological and pathological functions of Cdk5.
      • Liebl J.
      • Fürst R.
      • Vollmar A.M.
      • Zahler S.
      Twice switched at birth: cell cycle-independent roles of the “neuron-specific” cyclin-dependent kinase 5 (Cdk5) in non-neuronal cells.
      ).
      Under normal physiological conditions, CDK5 plays a pro-survival role as its inhibition by treatment with the somewhat selective inhibitor roscovitine, expression of a competitive-interfering loss of function mutant of CDK5 (CDK5-D145N) or siRNA-mediated knockdown results in cell dysfunction and cell death (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ). Substrates of CDK5 include transcription factors, kinases, cytoskeletal proteins, and apoptotic regulators. CDK5 directly phosphorylates NOXA (PMAIP1) (
      • Lowman X.H.
      • McDonnell M.A.
      • Kosloske A.
      • Odumade O.A.
      • Jenness C.
      • Karim C.B.
      • Jemmerson R.
      • Kelekar A.
      The proapoptotic function of Noxa in human leukemia cells is regulated by the kinase Cdk5 and by glucose.
      ) and HTRA2 (
      • Fitzgerald J.C.
      • Camprubi M.D.
      • Dunn L.
      • Wu H.C.
      • Ip N.Y.
      • Kruger R.
      • Martins L.M.
      • Wood N.W.
      • Plun-Favreau H.
      Phosphorylation of HtrA2 by cyclin-dependent kinase-5 is important for mitochondrial function.
      ) inhibiting their pro-apoptotic function and increases the anti-apoptotic activity of BCL2 by direct phosphorylation (
      • Cheung Z.H.
      • Gong K.
      • Ip N.Y.
      Cyclin-dependent kinase 5 supports neuronal survival through phosphorylation of Bcl-2.
      ) as well as indirectly increasing its expression (
      • Brinkkoetter P.T.
      • Wu J.S.
      • Ohse T.
      • Krofft R.D.
      • Schermer B.
      • Benzing T.
      • Pippin J.W.
      • Shankland S.J.
      p35, the non-cyclin activator of Cdk5, protects podocytes against apoptosis in vitro and in vivo.
      ). Under pathological conditions, such as Alzheimer disease, Parkinson disease, and Huntington disease, p35 can be cleaved by calpain proteases to generate a p25 fragment that has an extended half-life and lacks myristolation that induces hyper-activation and mis-location of CDK5 (
      • Shah K.
      • Lahiri D.K.
      Cdk5 activity in the brain - multiple paths of regulation.
      ,
      • Lopes J.P.
      • Agostinho P.
      Cdk5: multitasking between physiological and pathological conditions.
      ). Deregulation of CDK5 is also associated with cancer where it plays a role in regulating cell motility and metastasis (
      • Liu R.
      • Tian B.
      • Gearing M.
      • Hunter S.
      • Ye K.
      • Mao Z.
      Cdk5-mediated regulation of the PIKE-A-Akt pathway and glioblastoma cell invasion.
      ). Elevated expression of CDK5 correlates with poor prognosis in non-small-cell lung cancer (
      • Liu J.L.
      • Wang X.Y.
      • Huang B.X.
      • Zhu F.
      • Zhang R.G.
      • Wu G.
      Expression of CDK5/p35 in resected patients with non-small cell lung cancer: relation to prognosis.
      ) and the pharmacological inhibition of CDK5 as a strategy for cancer therapy has progressed to phase II clinical trials (
      • Arif A.
      Extraneuronal activities and regulatory mechanisms of the atypical cyclin-dependent kinase Cdk5.
      ).
      Protein damaging stress, including hyperthermia, can lead to apoptosis through BAX activation, mitochondrial release of cytochrome c, and caspase activation, although the molecular mechanisms responsible for BAX activation in heat-stressed cells are unclear (
      • Kennedy D.
      • Jäger R.
      • Mosser D.D.
      • Samali A.
      Regulation of apoptosis by heat shock proteins.
      ,
      • Mosser D.D.
      • Morimoto R.I.
      Molecular chaperones and the stress of oncogenesis.
      ). Sublethal exposure to hyperthermia induces the expression of molecular chaperone proteins, such as HSP70, which can inhibit apoptosis (
      • Mosser D.D.
      • Caron A.W.
      • Bourget L.
      • Denis-Larose C.
      • Massie B.
      Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.
      ,
      • Mosser D.D.
      • Martin L.H.
      Induced thermotolerance to apoptosis in a human T lymphocyte cell line.
      ). This pro-survival function of HSP70 contributes to the tumorigenic potential of cancer cells. As well, HSP70 plays a cytoprotective role in a number of neurodegenerative disorders by chaperoning the aggregation-prone proteins that are associated with these diseases (
      • Morimoto R.I.
      Proteotoxic stress and inducible chaperone networks in neurodegenerative disease and aging.
      ). We have previously shown that BAX activation in heat stressed cells is mediated by a NOXA-dependent depletion of the anti-apoptotic protein MCL1 (
      • Stankiewicz A.R.
      • Livingstone A.M.
      • Mohseni N.
      • Mosser D.D.
      Regulation of heat-induced apoptosis by Mcl-1 degradation and its inhibition by Hsp70.
      ). Since CDK5 has been shown to be a critical mediator of cell survival in stressed cells we investigated the effect of hyperthermia on CDK5 and examined whether its inhibition could increase the sensitivity of cells with elevated levels of HSP70 to hyperthermia.

      RESULTS

      We set out to determine whether hyperthermia affects CDK5 activity, given its role in regulating stress-induced apoptosis, and also to examine whether this might be affected by overexpression of HSP70, which has potent anti-apoptotic activity. For this we used PErTA70 cells, a human acute lymphoblastic T-cell line with tetracycline-regulated expression of HSP70 (
      • Mosser D.D.
      • Caron A.W.
      • Bourget L.
      • Meriin A.B.
      • Sherman M.Y.
      • Morimoto R.I.
      • Massie B.
      The chaperone function of hsp70 is required for protection against stress-induced apoptosis.
      ). Doxycycline induced cells, which overexpress HSP70 (+ HSP70) and non-induced cells (−HSP70) were exposed to 42, 43 or 44 °C for 1 h and then returned to 37 °C for 6 h. Western blotting showed a temperature dependent decrease in the abundance of both total and Tyr-15-phosphorylated CDK5 (pY15-CDK5) in Triton X-100 soluble extracts (Fig. 1A). Loss of pY15-CDK5 was more significant than that of total CDK5 in the heat-shocked cells. Overexpression of HSP70 suppressed the loss of total CDK5 and pY15-CDK5, particularly in cells exposed to 43 °C (Fig. 1B) and as shown previously (
      • Mosser D.D.
      • Caron A.W.
      • Bourget L.
      • Meriin A.B.
      • Sherman M.Y.
      • Morimoto R.I.
      • Massie B.
      The chaperone function of hsp70 is required for protection against stress-induced apoptosis.
      ) also inhibited caspase-3 activation (Fig. 1A). Levels of the CDK5 activator p35 were also decreased by hyperthermic treatment although not to the same extent as CDK5 or pY15-CDK5.
      Figure thumbnail gr1
      FIGURE 1Loss of pY15-CDK5 and insolubilization of total CDK5 in cells exposed to hyperthermia. A, PErTA70 cells that were either not induced (−HSP70) or induced to express HSP70 (+HSP70) were incubated at 42, 43, or 44 °C for 1 h and then returned to 37 °C for 6 h. Control cells remained at 37 °C. Cells were lysed in buffer containing 1% Triton X-100 and following centrifugation the Triton X-100 soluble fraction was examined by SDS-PAGE and Western blotting. B, quantification of the results shown in A (mean ± S.E., n = 3, * indicates a significant difference between −HSP70 and +HSP70 cells p < 0.05). C, cells were exposed to 42, 43, or 44 °C for 1 h and then either collected immediately or after incubation at 37 °C for 2, 4, or 6 h before collection. Cells were lysed as in A and following centrifugation the Triton X-100 soluble and insoluble (PELLET) fractions were examined by Western blotting.
      We next examined whether HSP70 expressing cells suffered less heat-inactivation of CDK5 and/or were able to recover soluble CDK5 and pY15-CDK5 more effectively when returned to 37 °C following heat shock. Cells were exposed to 42, 43, or 44 °C for 1 h and then either collected immediately or incubated at 37 °C for 2, 4 or 6 h before harvesting and lysis in Triton-X buffer (Fig. 1C). In the non-induced cells, pY15-CDK5 levels demonstrated mild recovery in cells heat-shocked at 42 °C and following 6 h of incubation at 37 °C, though no recovery of pY15-CDK5 was observed in cells exposed to either 43 or 44 °C. However, in HSP70-expressing cells complete recovery of pY15-CDK5 levels were observed by 2 h at 37 °C following exposure to 42 °C and a nearly complete return to basal levels occurred after 6 h of incubation following exposure to 43 °C. CDK5 was found in the Triton X-insoluble fractions of the non-induced cells following exposure to 42, 43 and 44 °C, while insoluble pY15-CDK5 was only present after exposure to 44 °C. In the HSP70-overexpressing cells CDK5 was observed in the insoluble fraction only after exposure to 44 °C and no appreciable level of pY15-CDK5 became insoluble at any temperature in these cells. These data demonstrate that total and pY15-CDK5 levels are sensitive to hyperthermia-induced loss. Furthermore, HSP70 overexpression enhances the recovery of pY15-CDK5 and prevents the insolubilization of total CDK5 following exposure to hyperthermia.
      We further explored the effect of hyperthermia on CDK5 solubility by exposing PErTA70 cells to 43 °C for various lengths of time followed by 6 h incubation at 37 °C (Fig. 2A). Levels of CDK5 and pY15-CDK5 were lost in a time-dependent manner following prolonged heat shock exposure in the non-induced cells though this loss was much less extreme in the HSP70-expressing cells (Fig. 2B). Loss of total CDK5 from the Triton X-soluble fraction was accompanied by its appearance in the insoluble fraction. However, pY15-CDK5 was not found in the insoluble fraction, indicating that hyperthermia leads to a rapid loss of CDK5 phosphorylation combined with the insolubilization of the pool of total CDK5. Probing for phosphorylated MAPK/CDK substrates revealed that hyperthermia caused an accumulation of high MW phosphoproteins in the insoluble fraction in the non-induced cells that did not occur in the HSP70-expressing cells (Fig. 2A). Heat shock also increased the level of phosphorylation of some proteins, notably proteins with a molecular size of ∼25 and 60 kDa. To test whether some of these proteins could potentially be targets of CDK5 we exposed cells to hyperthermia in the presence of the CDK5 inhibitor roscovitine (Fig. 2C). Inhibition of CDK5 in cells exposed to hyperthermia further decreased the level of pMAPK/CDK substrates and prevented the increased phosphorylation of the 25 and 60 kDa proteins observed in Fig. 2A. These results demonstrate that hyperthermia globally reduces the solubility and abundance of pMAPK/CDK substrates, including proteins phosphorylated by CDK5. As well, HSP70 has a protective effect in reducing the extent of pMAPK/CDK substrate insolubilization in heat-shocked cells.
      Figure thumbnail gr2
      FIGURE 2Effect of hyperthermia on phosphorylation of MAPK/CDK targets. A, non-induced (−HSP70) and induced (+HSP70) PErTA70 were exposed to 43 °C for 30, 60, 90, or 120 min and then incubated at 37 °C for 6 h before being lysed in 1% Triton X-100 buffer. Control cells were incubated at 37 °C (C). Triton X-100 soluble and insoluble (PELLET) fractions were examined by Western blotting. B, quantification of the results shown in A (mean ± S.E., n = 3, * indicates a significant difference between −HSP70 and +HSP70 cells p < 0.05). C, roscovitine enhances the loss of MAPK/CDK target protein phosphorylation in heat shocked cells and inhibits the hyperthermia-induced increase in phosphorylation of specific MAPK/CDK targets. PErTA70 cells were exposed to 43 °C for 60 or 120 min in the absence or presence of 20 μm roscovitine and collected. Roscovitine-treated cells were pre-incubated with the drug for 6 h at 37 °C before exposure to hyperthermia. Control cells were incubated at 37 °C. Cells were lysed with Triton X-100 lysis buffer, and the Triton X-100 soluble fractions were examined by Western blotting.
      Since exposure to mild hyperthermia (42 °C for 1 h) produced only a transient loss of pY15-CDK5 and had minimal effects on total CDK5 (Fig. 1C), we hypothesized that chemical inhibition of CDK5 activity using roscovitine would sensitize these cells to heat-induced apoptosis. To test this we exposed PErTA70 cells to 42 °C for 1 h either with or without roscovitine (5 or 10 μm) and then incubated them for 6 h at 37 °C, either without or with roscovitine, before measuring apoptosis by Annexin V staining (Fig. 3A). Treatment of both non-induced and HSP70-overexpressing cells with roscovitine at 37 °C produced a dose-dependent increase in apoptosis, which was further augmented by exposure to hyperthermia. However, overexpression of HSP70 lessened the effect of roscovitine on heat-induced apoptosis. We also looked at long-term survival following CDK5 inhibition in cells exposed to either 42 or 43 °C in the presence of various concentrations of roscovitine. Cells were heat shocked for 1 h in the presence of roscovitine and then incubated at 37 °C for 48 h in the continuous presence of roscovitine before measuring cell viability using the rezasurin reduction assay (Fig. 3B). The dose that reduced relative cell number by 50% (ED50) was similar in both the non-induced and HSP70-expressing cells treated at 37 °C (12 μm). However, when exposed to hyperthermia the non-induced cells were more sensitive to the combined effects of hyperthermia and roscovitine treatment (ED50, 6 μm) as compared with HSP70 overexpressing cells (ED, 10 μm). Therefore, inhibition of CDK5 activity with roscovitine effectively sensitized both non-induced and HSP70-overexpressing cells to hyperthermia, although HSP70 overexpression provided some protection against roscovitine-dependent heat sensitivity.
      Figure thumbnail gr3
      FIGURE 3The CDK5 inhibitor roscovitine sensitizes cells to heat-induced apoptosis. A, non-induced (−HSP70) and HSP70-expressing (+HSP70) cells were treated with 0, 5, or 10 μm roscovitine and incubated at either 37 °C for 7 h or exposed to 42 °C for 1 h followed by 6 h at 37 °C. Apoptosis was measured by Annexin-V staining (mean ± S.E., n = 3, * indicates significant difference in value compared with treatment in the absence of roscovitine p < 0.05). B, cells were incubated with various concentrations of roscovitine (0.5–20 μm), exposed to either 42 or 43 °C for 1 h and then incubated at 37 °C in the presence of roscovitine for 48 h. Viability was then measured using Alamar blue and plotted relative to cells that were not exposed to roscovitine (mean ± S.E., n = 3).
      We next examined whether the effect of CDK5 inhibition in PErTA70 cells exposed to mild hyperthermia correlated with an increased release of pro-apoptotic proteins from mitochondria (Fig. 4). In non-induced cells exposure to 42 °C caused the release of both cytochrome c and HtrA2 that was augmented in a dose-dependent manner by treatment with roscovitine, demonstrating that inhibition of CDK5 activity increases sensitivity to heat-induced apoptosis. Treatment of HSP70-overexpressing cells with 10 μm roscovitine that were exposed to 42 °C produced a significant increase in cytochrome c release although HtrA2 release was not affected by this combined treatment. Altogether these results show that inhibition of CDK5 with roscovitine sensitizes both non-induced and HSP70 overexpressing cells to hyperthermia-induced apoptosis.
      Figure thumbnail gr4
      FIGURE 4Roscovitine treatment augments the effects of hyperthermia on the release of cytochrome c and HtrA2 from mitochondria. A, cells were treated with 0, 5, or 10 μm roscovitine and incubated at 37 °C for 7 h or exposed to 42 °C for 1 h and then incubated at 37 °C for 6 h. Cells were lysed in 0.025% digitonin buffer and centrifugation to obtain membrane M and soluble S fractions, which were analyzed by Western blotting. Quantification of the results are shown for levels of cytochrome c (B) and HtrA2 (C), (mean ± S.E., n = 3, * indicates a significant difference between non-treated and roscovitine treated cells p < 0.05).
      The proapoptotic activity of NOXA is regulated by CDK5 under conditions of glucose deprivation (
      • Lowman X.H.
      • McDonnell M.A.
      • Kosloske A.
      • Odumade O.A.
      • Jenness C.
      • Karim C.B.
      • Jemmerson R.
      • Kelekar A.
      The proapoptotic function of Noxa in human leukemia cells is regulated by the kinase Cdk5 and by glucose.
      ). We were therefore interested in determining whether inhibition of CDK5 by roscovitine treatment would augment the effect of mild hyperthermia on NOXA and MCL1 protein levels. PErTA70 cells were treated with either 5 or 10 μm roscovitine and exposed to 42 °C for 1 h followed by incubation at 37 °C for 6 h (Fig. 5A). NOXA levels were increased to a similar extent in both non-induced and HSP70-overexpressing cells when treated with 10 μm roscovitine at 37 °C. When exposed to hyperthermia NOXA levels were elevated in non-induced cells but not in HSP70-overexpressing cells. However, exposure to hyperthermia in the presence of roscovitine resulted in an increased abundance of NOXA in the HSP70-overexpressing cells and further increased the levels of NOXA protein in the non-induced cells (Fig. 5B). This increased abundance of NOXA correlated with a significant decrease in the levels of MCL1 in both the absence and presence of HSP70 (Fig. 5C). The pro-apoptotic function of NOXA is mediated in part by its ability to target MCL1 for proteasomal degradation (
      • Ploner C.
      • Kofler R.
      • Villunger A.
      Noxa: at the tip of the balance between life and death.
      ).
      Figure thumbnail gr5
      FIGURE 5Roscovitine treatment augments the effects of hyperthermia on NOXA accumulation. A, cells were treated with 0, 5, or 10 μm roscovitine and incubated at 37 °C for 7 h or exposed to 42 °C for 1 h and then incubated at 37 °C for 6 h. Cells were then lysed in Laemmli buffer and analyzed by Western blotting. Quantification of the results are shown for levels of NOXA (B) and MCL1 (C), (mean ± S.E., n = 3, * indicates a significant difference between non-treated and roscovitine-treated cells, p < 0.05).
      We next sought to address the mechanism responsible for the increased abundance of NOXA in the roscovitine-treated cells. We considered the possibility that inhibition of CDK5 activity could reduce NOXA phosphorylation, resulting in a reduced rate of protein turnover and a corresponding increase in protein abundance. For these experiments we used PEER cells (
      • Ravid Z.
      • Goldblum N.
      • Zaizov R.
      • Schlesinger M.
      • Kertes T.
      • Minowada J.
      • Verbi W.
      • Greaves M.
      Establishment and characterization of a new leukaemic T-cell line (Peer) with an unusual phenotype.
      ), which were used previously as the parental line to create stable PErTA70 cells. PEER cells were incubated with 10 μm roscovitine for 6 h at 37 °C and subsequently treated with cycloheximide for up to 120 min to prevent further NOXA protein synthesis. Cells were collected at various times following cycloheximide addition, and NOXA protein turnover rate was measured in the control and roscovitine-treated cells (Fig. 6A). Surprisingly, although the roscovitine-treated cells had accumulated higher levels of NOXA protein than the non-treated cells the turnover rates were identical. To rule out potential off-target effects of roscovitine we produced a stably-transfected PEER cell line with tetracycline regulated expression of a competitive-interfering loss of function mutant of CDK5 (CDK5-D145N) that is kinetically dead and competes with wild-type CDK5 for binding of the CDK5 activator p35 (
      • van den Heuvel S.
      • Harlow E.
      Distinct roles for cyclin-dependent kinases in cell cycle control.
      ). Doxycycline induced expression of CDK5-D145N resulted in an increase in NOXA protein levels, though again this was not due to an effect on NOXA protein turnover since the rate of turnover was unchanged between non-induced and CDK5-D145N expressing cells (Fig. 6B). It has been previously demonstrated that NOXA is phosphorylated on residue serine-13 by CDK5 and that this lessens its pro-apoptotic activity (
      • Lowman X.H.
      • McDonnell M.A.
      • Kosloske A.
      • Odumade O.A.
      • Jenness C.
      • Karim C.B.
      • Jemmerson R.
      • Kelekar A.
      The proapoptotic function of Noxa in human leukemia cells is regulated by the kinase Cdk5 and by glucose.
      ). Therefore, we examined the turnover rate of NOXA in transiently transfected HEK-293-rtTA cells expressing either wild-type NOXA or a non-phosphorylatable Ser-13-to-alanine (S13A) mutant. Examination of NOXA protein turnover in these cells showed that prevention of NOXA serine 13 phosphorylation did not alter the turnover rate of NOXA (Fig. 6C). Taken together, these results demonstrate that phosphorylation of NOXA by CDK5 does not alter NOXA protein turnover and therefore CDK5 must affect NOXA expression by controlling its rate of synthesis.
      Figure thumbnail gr6
      FIGURE 6CDK5 does not regulate NOXA protein half-life. A, PEER cells were incubated in the absence or presence of 10 μm roscovitine for 6 h and then either collected immediately or incubated with cycloheximide for 30, 60, 90, or 120 min before collection and analysis of NOXA protein levels by Western blotting. B, PErTA cells with tetracycline-regulated expression of a competitive-interfering loss of function mutant of CDK5-D145N (CDK5-DN) were incubated without doxycycline (−CDK5-DN) or with doxycycline (+CDK5-DN) for 24 h and then either collected immediately or incubated with cycloheximide for 30, 60, 90, or 120 min before collection and analysis of NOXA protein levels by Western blotting. C, 293-rtTA cells were transiently transfected with plasmids to express wild-type (WT) NOXA or NOXA with a serine-13-to-alanine mutation (S13A). Following transfection the cells were either collected immediately or incubated with cycloheximide for 40, 80, or 120 min before collection and analysis of NOXA protein levels by Western blotting.
      We recently demonstrated that NOXA expression is regulated by the microRNA miR-23a (
      • Roufayel R.
      • Johnston D.S.
      • Mosser D.D.
      The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.
      ). Exposure to hyperthermia reduces miR-23a levels resulting in increased accumulation of NOXA mRNA and protein. We therefore considered that CDK5 inhibition could increase NOXA expression by altering the expression of miR-23a. We used RT-qPCR to measure miR-23a and NOXA mRNA levels in roscovitine treated PEER cells and found that treatment with 10 μm roscovitine for 6 h at 37 °C resulted in a 30-fold reduction in miR-23a levels and a greater than 20-fold increase in the levels of NOXA mRNA (Fig. 7A). Similar results were seen in cells expressing CDK5-D145N demonstrating that this effect is specific to CDK5 inhibition (Fig. 7B). To test whether the ability of CDK5-D145N to reduce miR-23a levels could be rescued by restoring CDK5 activity, we expressed CDK5-D145N in HeLa cells with increasing amounts of the CDK5 activator p35 (Fig. 7C). Overexpression of CDK5-D145N reduced miR-23a levels nearly 30-fold (Fig. 7D). However, transfection with a 2.5-fold excess of the p35 expression plasmid over the CDK5-D145N expression plasmid restored miR-23a levels. Restoration of miR-23a levels correlated with a decrease in the abundance of NOXA mRNA. The levels of miR-23a where further increased while NOXA mRNA were further decreased when the amount of the p35 expression plasmid was increased to 5- or 10-fold excess over that of the CDK5-D145N expression plasmid. Together these results suggest that CDK5 activity negatively regulates NOXA mRNA expression, and subsequent protein levels, by positively regulating miR-23a expression.
      Figure thumbnail gr7
      FIGURE 7CDK5 inhibition reduces miR-23a levels causing increased NOXA expression. A, PEER cells were incubated without or with 10 μm roscovitine for 6 h and then analyzed for miR-23a and NOXA mRNA levels by RT-qPCR (mean ± S.E., n = 3). Results of semi-quantitative RT-PCR are shown on the right. B, stably-transfected PErTA cells with tetracycline-regulated expression of CDK5-D145N (CDK5-DN) were incubated without doxycycline (−CDK5-DN) or with doxycycline (+CDK5-DN) for 24 h and then analyzed for miR-23a and NOXA mRNA levels by RT-qPCR (mean ± S.E., n = 3) and semi-quantitative RT-PCR. C, Western blots showing levels of overexpressed p35 and CDK5-D145N proteins in transfected HeLa cells. Cells were transiently transfected with a plasmid to overexpress CDK5-D145N (2.5 μg) without or with a 2.5-, 5-, or 10-fold excess of a p35 expression plasmid. Cells were collected 24 h post-transfection. D, cells transfected as in C were analyzed for miR-23a and NOXA mRNA levels by RT-qPCR (mean ± S.E., n = 3).
      We lastly examined the effect of miR-23a overexpression on resistance to apoptosis in cells exposed to hyperthermia in the presence and absence of roscovitine. Stably transfected PEER cells overexpressing miR-23a produced lower levels of NOXA mRNA relative to stably transfected cells expressing a control miRNA (Fig. 8A). Roscovitine treatment of the control miRNA-expressing cells resulted in increased NOXA protein levels and caspase-3 cleavage (Fig. 8B). The extent of NOXA protein accumulation and caspase-3 cleavage was higher when these cells were exposed to hyperthermia (42 °C) while in the presence of roscovitine. Remarkably the miR-23a-expressing cells showed no increase in NOXA protein levels or caspase-3 cleavage when treated with roscovitine either alone or in combination with hyperthermia. Additionally, while roscovitine treatment of the control miRNA expressing cells resulted in apoptosis, as indicated by the appearance of cleaved caspase-3 (Fig. 8B) and by an increased percentage of Annexin V positive cells (Fig. 8C), the miR-23a expressing cells were highly resistant to the combined treatment of roscovitine with hyperthermia (Fig. 8, B and C). Therefore, the effects of CDK5 inhibition on sensitivity to hyperthermia-induced apoptosis can be abrogated by the overexpression of miR-23a and its ability to suppress NOXA expression.
      Figure thumbnail gr8
      FIGURE 8Overexpression of miR-23a prevents NOXA expression in cells treated with mild heat shock or roscovitine. A, RT-qPCR analysis of miR-23a and NOXA mRNA levels in PEER cells that stably overexpress a control miRNA (C-miR) or miR-23a (mean ± S.E., n = 3). Results of semi-quantitative RT-PCR are shown on the right. B, control miRNA and miR-23a-overexpressing cells were incubated with 0 or 10 μm roscovitine and incubated at either 37 °C for 7 h or exposed to 42 °C for 1 h and then incubated at 37 °C for 6 h. NOXA and active caspase-3 (p17) levels were measured by Western blotting. C, measurement of apoptosis by Annexin-V staining of samples treated as described in B (mean ± S.E., n = 3, * indicates a significant difference between non-treated and roscovitine-treated cells p < 0.05).

      DISCUSSION

      Proteotoxic stress, including hyperthermic exposure, causes protein misfolding and aggregation leading to loss of function. Signaling pathways controlling cell survival are acutely affected by proteotoxic stress. Hyperthermia can either activate or inhibit signaling pathways by inhibiting the activity of specific kinases or the phosphatases that regulate their activity (
      • Mosser D.D.
      • Caron A.W.
      • Bourget L.
      • Denis-Larose C.
      • Massie B.
      Role of the human heat shock protein hsp70 in protection against stress-induced apoptosis.
      ,
      • Meriin A.B.
      • Yaglom J.A.
      • Gabai V.L.
      • Zon L.
      • Ganiatsas S.
      • Mosser D.D.
      • Zon L.
      • Sherman M.Y.
      Protein-damaging stresses activate c-Jun N-terminal kinase via inhibition of its dephosphorylation: a novel pathway controlled by HSP72.
      ,
      • Yaglom J.
      • O'Callaghan-Sunol C.
      • Gabai V.
      • Sherman M.Y.
      Inactivation of dual-specificity phosphatases is involved in the regulation of extracellular signal-regulated kinases by heat shock and hsp72.
      • Simard J.P.
      • Reynolds D.N.
      • Kraguljac A.P.
      • Smith G.S.
      • Mosser D.D.
      Overexpression of HSP70 inhibits cofilin phosphorylation and promotes lymphocyte migration in heat-stressed cells.
      ). Induction of apoptosis in cells exposed to hyperthermic stress serves as a mechanism to remove irreparably damaged cells. However, prior exposure to a mild proteotoxic stress induces an adaptive response leading to the enhanced expression of the heat-shock family of molecular chaperones, including HSP70, which have anti-apoptotic properties (
      • Mosser D.D.
      • Morimoto R.I.
      Molecular chaperones and the stress of oncogenesis.
      ). HSP70 inhibits apoptosis by preventing the activation of BAX, a pro-apoptotic member of the BCL2 family of apoptotic regulators (
      • Stankiewicz A.R.
      • Lachapelle G.
      • Foo C.P.
      • Radicioni S.M.
      • Mosser D.D.
      Hsp70 inhibits heat-induced apoptosis upstream of mitochondria by preventing Bax translocation.
      ). The activation of BAX in heat-stressed cells is controlled by a NOXA-dependent loss of the anti-apoptotic BCL2 family member MCL1 (
      • Stankiewicz A.R.
      • Livingstone A.M.
      • Mohseni N.
      • Mosser D.D.
      Regulation of heat-induced apoptosis by Mcl-1 degradation and its inhibition by Hsp70.
      ).
      The proapoptotic activity of NOXA is regulated by CDK5 dependent phosphorylation of residue serine 13 (
      • Lowman X.H.
      • McDonnell M.A.
      • Kosloske A.
      • Odumade O.A.
      • Jenness C.
      • Karim C.B.
      • Jemmerson R.
      • Kelekar A.
      The proapoptotic function of Noxa in human leukemia cells is regulated by the kinase Cdk5 and by glucose.
      ). Lymphoid cells deprived of glucose undergo apoptosis that is associated with reduced CDK5-mediated NOXA serine 13 phosphorylation, increased NOXA/MCL1 association and MCL1 depletion (
      • Lowman X.H.
      • McDonnell M.A.
      • Kosloske A.
      • Odumade O.A.
      • Jenness C.
      • Karim C.B.
      • Jemmerson R.
      • Kelekar A.
      The proapoptotic function of Noxa in human leukemia cells is regulated by the kinase Cdk5 and by glucose.
      ). We were therefore interested in examining the potential effect of hyperthermia on CDK5 activity and its relation to NOXA-induced heat-sensitivity. We found that CDK5 is sensitive to heat-induced insolubilization and that HSP70 overexpression maintains CDK5 solubility in heat-stressed cells. We also observed a dramatic loss of tyrosine-15 phosphorylated CDK5 after exposure to hyperthermia, which was prevented by HSP70 overexpression. Previously, several studies have shown that the kinase activity of CDK5 is stimulated by Tyr-15 phosphorylation and that this is mediated by c-Abl, Fyn, and Src kinases (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ). One study investigating c-Abl has shown that hyperthermia reduces c-Abl mRNA expression in vivo in mice testes (
      • Rockett J.C.
      • Mapp F.L.
      • Garges J.B.
      • Luft J.C.
      • Mori C.
      • Dix D.J.
      Effects of hyperthermia on spermatogenesis, apoptosis, gene expression, and fertility in adult male mice.
      ), while another study demonstrated that hyperthermia reduces activity of purified recombinant c-Abl (
      • Jain S.K.
      • de Aos I.
      • Inai Y.
      • Liu F.
      • Varticovski L.
      Inactivation of wild-type BCR/ABL tyrosine kinase in hematopoietic cells by mild hyperthermia.
      ). In addition, Gao et al., (
      • Gao C.
      • Negash S.
      • Wang H.S.
      • Ledee D.
      • Guo H.
      • Russell P.
      • Zelenka P.
      Cdk5 mediates changes in morphology and promotes apoptosis of astrocytoma cells in response to heat shock.
      ) have reported heat-induced insolubilization of CDK5 in astrocytes and showed that overexpression of CDK5 protected these cells from the effects of hyperthermia. Therefore, in combination with previous studies, our results suggest that CDK5 activity is impaired during hyperthermia, and that this loss of activity could contribute to hyperthermia-induced apoptosis through decreased substrate phosphorylation, such as NOXA. Lastly, we hypothesize that HSP70 could potentially prevent hyperthermia-induced apoptosis by maintaining CDK5 activity through preservation of CDK5 solubility and/or Tyr-15 phosphorylation via maintenance of c-Abl.
      Though Tyr-15 phosphorylation of CDK5 has been shown to increase kinase activity, a recent report casts doubt on the role of Tyr-15 phosphorylation in the regulation of CDK5 activity (
      • Kobayashi H.
      • Saito T.
      • Sato K.
      • Furusawa K.
      • Hosokawa T.
      • Tsutsumi K.
      • Asada A.
      • Kamada S.
      • Ohshima T.
      • Hisanaga S.
      Phosphorylation of cyclin-dependent kinase 5 (Cdk5) at Tyr-15 is inhibited by Cdk5 activators and does not contribute to the activation of Cdk5.
      ). We therefore also examined the effect of CDK5 inhibition on heat sensitivity using the CDK5 inhibitor roscovitine. Given that cells exposed to 42 °C were able to recover total CDK5 and pY15-CDK5 levels when allowed to recover at 37 °C post-hyperthermia and had only minimal levels of active caspase-3, we hypothesized that inhibition of CDK5 with roscovitine should sensitize cells to mild hyperthermia. As anticipated, roscovitine treatment increased cytochrome c release and apoptosis in cells exposed to 42 °C, corresponding with an increased abundance of NOXA protein and a loss of MCL1.
      Roscovitine is a potent inhibitor of CDK5 (IC50 of 0.2 μm) as well as CDK1 and CDK2 (IC50 of 0.7 μm) but has minimal inhibitory activity against a number of other kinases (
      • Meijer L.
      • Borgne A.
      • Mulner O.
      • Chong J.P.
      • Blow J.J.
      • Inagaki N.
      • Inagaki M.
      • Delcros J.G.
      • Moulinoux J.P.
      Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5.
      ). Roscovitine treatment has been reported to induce apoptosis that was associated with decreased levels of MCL1 in neutrophils and multiple myeloma cells (
      • MacCallum D.E.
      • Melville J.
      • Frame S.
      • Watt K.
      • Anderson S.
      • Gianella-Borradori A.
      • Lane D.P.
      • Green S.R.
      Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1.
      ,
      • Raje N.
      • Kumar S.
      • Hideshima T.
      • Roccaro A.
      • Ishitsuka K.
      • Yasui H.
      • Shiraishi N.
      • Chauhan D.
      • Munshi N.C.
      • Green S.R.
      • Anderson K.C.
      Seliciclib (CYC202 or R-roscovitine), a small-molecule cyclin-dependent kinase inhibitor, mediates activity via down-regulation of Mcl-1 in multiple myeloma.
      • Gautam S.
      • Kirschnek S.
      • Wiesmeier M.
      • Vier J.
      • Häcker G.
      Roscovitine-induced apoptosis in neutrophils and neutrophil progenitors is regulated by the Bcl-2-family members Bim, Puma, Noxa and Mcl-1.
      ). Interestingly, Gautam et al., (
      • Gautam S.
      • Kirschnek S.
      • Wiesmeier M.
      • Vier J.
      • Häcker G.
      Roscovitine-induced apoptosis in neutrophils and neutrophil progenitors is regulated by the Bcl-2-family members Bim, Puma, Noxa and Mcl-1.
      ) observed an increased abundance of NOXA mRNA in roscovitine-treated neutrophils, although protein levels were not measured. We found that both hyperthermic exposure and roscovitine treatment on their own caused a significant increase in NOXA protein and that the combined treatment of hyperthermia plus roscovitine augmented this increase. This led us to speculate that CDK5 activity might play a role in regulating the half-life of NOXA such that CDK5 inhibition, by heat shock or roscovitine treatment, could result in decreased NOXA phosphorylation and a reduced rate of turnover. However, we found that neither roscovitine treatment, overexpression of CDK5-D145N or prevention of NOXA phosphorylation by a Ser-13-alanine mutation (S13A) had any effect on the turnover rate of NOXA. Interestingly treatment of cells with roscovitine or overexpression of CDK5-D145N resulted in an increased abundance of NOXA protein suggesting that CDK5 inhibition directly affects NOXA expression.
      The observation that CDK5 inhibition caused an increased accumulation of NOXA protein without affecting its turnover rate led us to consider that NOXA expression was being affected at the level of mRNA expression or translation. We have recently found that NOXA expression is controlled by the microRNA miR-23a (
      • Roufayel R.
      • Johnston D.S.
      • Mosser D.D.
      The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.
      ), whereby mir-23a binding to NOXA mRNA affects both transcript levels and translation. We have also demonstrated that hyperthermia causes a reduction in miR-23a levels, which results in an increased abundance of NOXA mRNA and protein leading to cell death (
      • Roufayel R.
      • Johnston D.S.
      • Mosser D.D.
      The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.
      ). Consequently, we hypothesized that miR-23a levels might be regulated by CDK5. As predicted, our results demonstrate that either roscovitine treatment or expression of CDK5-D145N caused a dramatic reduction in miR-23a levels and a corresponding increase in NOXA mRNA. In the case of CDK5-D145N expression, the loss of miR-23a could be rescued by overexpression of the CDK5 activator p35 resulting in a corresponding decrease in Noxa mRNA expression. Additionally, overexpression of miR-23a provided resistance to apoptosis in cells exposed to roscovitine alone or in combination with hyperthermia most likely through depletion of NOXA mRNA.
      Given that inhibition of CDK5 activity reduces miR-23a expression, we speculate that CDK5 regulates the activity of a transcription factor controlling miR-23a expression. A number of transcription factors have been implicated in the regulation of miR-23a expression including Srf, Myf6, NFATc3; c-myc and CREB (
      • Hernandez-Torres F.
      • Aranega A.E.
      • Franco D.
      Identification of regulatory elements directing miR-23a-miR-27a-miR-24–2 transcriptional regulation in response to muscle hypertrophic stimuli.
      • Lin Z.
      • Murtaza I.
      • Wang K.
      • Jiao J.
      • Gao J.
      • Li P.F.
      miR-23a functions downstream of NFATc3 to regulate cardiac hypertrophy.
      ,
      • Li X.
      • Liu X.
      • Xu W.
      • Zhou P.
      • Gao P.
      • Jiang S.
      • Lobie P.E.
      • Zhu T.
      c-MYC-regulated miR-23a/24–2/27a cluster promotes mammary carcinoma cell invasion and hepatic metastasis by targeting Sprouty2.
      • Tan X.
      • Wang S.
      • Zhu L.
      • Wu C.
      • Yin B.
      • Zhao J.
      • Yuan J.
      • Qiang B.
      • Peng X.
      cAMP response element-binding protein promotes gliomagenesis by modulating the expression of oncogenic microRNA-23a.
      ), though it is currently unknown if these transcription factors are altered during hyperthermia with the exception of c-myc (
      • Bukh A.
      • Martinez-Valdez H.
      • Freedman S.J.
      • Freedman M.H.
      • Cohen A.
      The expression of c-fos, c-jun, and c-myc genes is regulated by heat shock in human lymphoid cells.
      ). Alternatively, both hyperthermia and roscovitine treatment are known to inhibit RNA polymerase II, which could potentially affect miR-23a levels. MacCallum et al., (
      • MacCallum D.E.
      • Melville J.
      • Frame S.
      • Watt K.
      • Anderson S.
      • Gianella-Borradori A.
      • Lane D.P.
      • Green S.R.
      Seliciclib (CYC202, R-Roscovitine) induces cell death in multiple myeloma cells by inhibition of RNA polymerase II-dependent transcription and down-regulation of Mcl-1.
      ) suggest that roscovitine induces apoptosis through inhibition of RNA polymerase II dependent transcription, which would result in the selective loss of short-lived proteins such as MCL1. MiR-23a is transcribed as part of a pri-miRNA cluster by RNA polymerase II (
      • Lee Y.
      • Kim M.
      • Han J.
      • Yeom K.H.
      • Lee S.
      • Baek S.H.
      • Kim V.N.
      MicroRNA genes are transcribed by RNA polymerase II.
      ) and therefore hyperthermia or roscovitine treatment could potentially reduce miR-23a levels by directly inhibiting RNA polymerase II, thereby causing an increase in NOXA mRNA and protein abundance. However, NOXA, like MCL1, also has a short protein half-life and therefore it is unlikely that inhibition of RNA polymerase II would cause an increase in NOXA protein levels, suggesting that inhibition of CDK5 activity and the resulting reduced expression of miR-23a are the definitive factors controlling NOXA expression during hyperthermia.
      MicroRNAs are important regulators of cell survival and not surprisingly their expression is often deregulated in a number of diseases including cancer (
      • Lima R.T.
      • Busacca S.
      • Almeida G.M.
      • Gaudino G.
      • Fennell D.A.
      • Vasconcelos M.H.
      MicroRNA regulation of core apoptosis pathways in cancer.
      ). MiR-23a is transcribed as part of the miR-23a∼27a∼24–2 cluster, which is overexpressed in and is implicated in the pathology of cancer and cardiac hypertrophy (
      • Chhabra R.
      • Dubey R.
      • Saini N.
      Cooperative and individualistic functions of the microRNAs in the miR-23a∼27a∼24–2 cluster and its implication in human diseases.
      ). It acts as a pro-survival factor by suppressing the expression of the pro-apoptotic regulators APAF1, caspase-7 and NOXA (
      • Roufayel R.
      • Johnston D.S.
      • Mosser D.D.
      The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.
      ,
      • Chen Q.
      • Xu J.
      • Li L.
      • Li H.
      • Mao S.
      • Zhang F.
      • Zen K.
      • Zhang C.Y.
      • Zhang Q.
      MicroRNA-23a/b and microRNA-27a/b suppress Apaf-1 protein and alleviate hypoxia-induced neuronal apoptosis.
      ,
      • Mao J.
      • Lv Z.
      • Zhuang Y.
      MicroRNA-23a is involved in tumor necrosis factor-alpha induced apoptosis in mesenchymal stem cells and myocardial infarction.
      ). There are only a few reports on the effects of hyperthermia on microRNA expression (
      • Place R.F.
      • Noonan E.J.
      Non-coding RNAs turn up the heat: an emerging layer of novel regulators in the mammalian heat shock response.
      ). We have previously shown that hyperthermia causes miR-23a levels to decrease and that this results in the increased expression of NOXA leading to apoptosis (
      • Roufayel R.
      • Johnston D.S.
      • Mosser D.D.
      The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.
      ). The results presented here suggest that the decreased abundance of miR-23a in heat-shocked cells is due to CDK5 inhibition. HSP70 overexpression prevents the heat-induced loss of miR-23a (
      • Roufayel R.
      • Johnston D.S.
      • Mosser D.D.
      The elimination of miR-23a in heat-stressed cells promotes NOXA-induced cell death and is prevented by HSP70.
      ), which we suggest could be attributed to the ability of HSP70 to prevent the heat-induced insolubilization of total CDK5 and loss of pY15-CDK5.
      CDK5 plays a critical role in regulating a number of vital cellular processes including cell, survival and migration in a variety of cell types, including lymphoid cells (
      • Hisanaga S.
      • Endo R.
      Regulation and role of cyclin-dependent kinase activity in neuronal survival and death.
      ,
      • Arif A.
      Extraneuronal activities and regulatory mechanisms of the atypical cyclin-dependent kinase Cdk5.
      ,
      • Contreras-Vallejos E.
      • Utreras E.
      • Gonzalez-Billault C.
      Going out of the brain: non-nervous system physiological and pathological functions of Cdk5.
      • Liebl J.
      • Fürst R.
      • Vollmar A.M.
      • Zahler S.
      Twice switched at birth: cell cycle-independent roles of the “neuron-specific” cyclin-dependent kinase 5 (Cdk5) in non-neuronal cells.
      ). Deregulation of CDK5 activity is associated with the pathology of cancer, inflammation, diabetes and protein folding diseases including Alzheimer disease, amyotrophic lateral sclerosis, and Parkinson disease. Our results suggest that cellular stress could contribute to disease pathology through suppression of CDK5 activity and the subsequent effects on miR-23a and NOXA expression. CDK5 activity is often up-regulated in many cancers, potentially leading to decreased NOXA expression and protection against stress-induced apoptosis. Currently there are multiple CDK5 inhibitors that are being investigated both in vitro and in clinical trials that have shown efficacy against CDK5 (
      • Demange L.
      • Abdellah F.N.
      • Lozach O.
      • Ferandin Y.
      • Gresh N.
      • Meijer L.
      • Galons H.
      Potent inhibitors of CDK5 derived from roscovitine: synthesis, biological evaluation and molecular modelling.
      • Fabre C.
      • Gobbi M.
      • Ezzili C.
      • Zoubir M.
      • Sablin M.P.
      • Small K.
      • Im E.
      • Shinwari N.
      • Zhang D.
      • Zhou H.
      • Le Tourneau C.
      Clinical study of the novel cyclin-dependent kinase inhibitor dinaciclib in combination with rituximab in relapsed/refractory chronic lymphocytic leukemia patients.
      ,
      • Lapenna S.
      • Giordano A.
      Cell cycle kinases as therapeutic targets for cancer.
      • Stephenson J.J.
      • Nemunaitis J.
      • Joy A.A.
      • Martin J.C.
      • Jou Y.M.
      • Zhang D.
      • Statkevich P.
      • Yao S.L.
      • Zhu Y.
      • Zhou H.
      • Small K.
      • Bannerji R.
      • Edelman M.J.
      Randomized phase 2 study of the cyclin-dependent kinase inhibitor dinaciclib (MK-7965) versus erlotinib in patients with non-small cell lung cancer.
      ).
      A major finding of this study is that the cytoprotective properties of HSP70 can be attributed in part to its ability to prevent CDK5 inactivation in stressed cells. HSP70 is often overexpressed in tumor cells where it acts as an inhibitor of apoptosis and contributes to the process of tumorigenesis (
      • Mosser D.D.
      • Morimoto R.I.
      Molecular chaperones and the stress of oncogenesis.
      ). We hypothesize that HSP70 might allow cancer cells to endure the oxidative and proteotoxic tumor microenvironment by helping to maintain the activity of CDK5. In support of this we have demonstrated the cytoprotective effect of HSP70 overexpression on both CDK5 solubility and Tyr-15 phosphorylation during hyperthermia, which ultimately results in reduced NOXA protein expression and protection against hyperthermia-induced apoptosis. Overall our results suggest that a combination therapy of hyperthermia with CDK5 inhibitors could be effective in the treatment of cancers that have elevated levels of HSP70.

      Acknowledgments

      We thank Jing Zhang from the University of Guelph Genomics Facility for assistance with the qRT-PCR analysis.

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