Dexmedetomidine ameliorates high glucose-induced epithelial-mesenchymal transformation in HK-2 cells through the Cdk5/Drp1/ROS pathway.

Epithelial-mesenchymal transformation (EMT) plays an important role in the progression of diabetic nephropathy. Dexmedetomidine (DEX) has shown renoprotective effects against ischemic reperfusion injury; however, whether and how DEX prevents high glucose-induced EMT in renal tubular epithelial cells is incompletely known. Here, we conduct in vitro experiments using HK-2 cells, a human tubular epithelial cell line. Our results demonstrate that high glucose increases the expressions of EMT-related proteins, including Vimentin, Slug, Snail and Twist, while decreasing the expression of E-cadherin and increasing Cdk5 expression in HK-2 cells. Both Cdk5 knockdown and inhibition by roscovitine increase the expressions of E-cadherin while decreasing the expressions of other EMT-related markers. DEX inhibits Cdk5 expression without affecting cell viability and changes the expressions of EMT-related markers, similar to effects of Cdk5 inhibition. Furthermore, Cdk5 is found to interact with Drp1 at the protein level and mediate the phosphorylation of Drp1. In addition, Drp1 inhibition with mdivi-1 could also restrain the high glucose-induced EMT process in HK-2 cells. Immunofluorescence results show that roscovitine, Mdivi-1 and DEX inhibit high glucose-induced intracellular ROS accumulation, while the oxidant H 2O 2 eliminates the protective effect of DEX on the EMT process. These results indicate that DEX mitigates high glucose-induced EMT progression in HK-2 cells via inhibition of the Cdk5/Drp1/ROS pathway.

Ubiquitin-specific protease 8 inhibitor suppresses adrenocorticotropic hormone production and corticotroph tumor cell proliferation.

Cushing's disease is primarily caused by autonomic hypersecretion of adrenocorticotropic hormone (ACTH) from a pituitary adenoma. In Cushing's disease, mutations in the ubiquitin-specific protease 8 (USP8) have been detected. These mutations are associated with hyperactivation of USP8 that prevent epidermal growth factor receptor (EGFR) degradation. This leads to increased EGFR stability and results in the maintenance of EGFR signaling in Cushing's disease. USP8 inhibitors can suppress the growth of various tumors. In this study, the effects of a potent USP8 inhibitor, DUBs-IN-2, on ACTH production and cell proliferation were examined in mouse corticotroph tumor (AtT-20) cells. Proopiomelanocortin (Pomc) mRNA levels and ACTH levels were decreased in AtT-20 cells by DUBs-IN-2. Further, cell proliferation was inhibited, and apoptosis was induced by DUBs-IN-2. Transcript levels of pituitary tumor-transforming gene 1 (Pttg1), a pituitary tumor growth marker, were increased; and transcript levels of stress response growth arrest and DNA damage-inducible 45 (Gadd45ß) and Cdk5 and ABL enzyme substrate 1 (Cables1) mRNA levels were increased in response to the drug. Gadd45ß or Cables1 knockdown partially inhibited the DUBs-IN-2-induced decrease in cell proliferation, but not Pomc mRNA levels. Both GADD45ß and CABLES1 may be responsible, at least in part, for the USP8-induced suppression of corticotroph tumor cell proliferation. USP-8 may be a new treatment target in Cushing's disease.

CB1R regulates CDK5 signaling and epigenetically controls Rac1 expression contributing to neurobehavioral abnormalities in mice postnatally exposed to ethanol.

Fetal alcohol spectrum disorders (FASD) represent a wide array of defects that arise from ethanol exposure during development. However, the underlying molecular mechanisms are limited. In the current report, we aimed to further evaluate the cannabinoid receptor type 1 (CB1R)-mediated mechanisms in a postnatal ethanol-exposed animal model. We report that the exposure of postnatal day 7 (P7) mice to ethanol generates p25, a CDK5-activating peptide, in a time- and CB1R-dependent manner in the hippocampus and neocortex brain regions. Pharmacological inhibition of CDK5 activity before ethanol exposure prevented accumulation of cleaved caspase-3 (CC3) and hyperphosphorylated tau (PHF1) (a marker for neurodegeneration) in neonatal mice and reversed cAMP response element-binding protein (CREB) activation and activity-regulated cytoskeleton-associated protein (Arc) expression. We also found that postnatal ethanol exposure caused a loss of RhoGTPase-related, Rac1, gene expression in a CB1R and CDK5 activity-dependent manner, which persisted to adulthood. Our epigenetic analysis of the Rac1 gene promoter suggested that persistent suppression of Rac1 expression is mediated by enhanced histone H3 lysine 9 dimethylation (H3K9me2), a repressive chromatin state, via G9a recruitment. The inhibition of CDK5/p25 activity before postnatal ethanol exposure rescued CREB activation, Arc, chromatin remodeling and Rac1 expression, spatial memory, and long-term potentiation (LTP) abnormalities in adult mice. Together, these findings propose that the postnatal ethanol-induced CB1R-mediated activation of CDK5 suppresses Arc and Rac1 expression in the mouse brain and is responsible for persistent synaptic plasticity and learning and memory defects in adult mice. This CB1R-mediated activation of CDK5 signaling during active synaptic development may slow down the maturation of synaptic circuits and may cause neurobehavioral defects, as found in this FASD animal model.

CDK5 Regulates PD-L1 Expression and Cell Maturation in Dendritic Cells of CRSwNP.

The maturation of dendritic cells is critical for chronic rhinosinusitis with nasal polyps (CRSwNPs), especially eosinophilic chronic rhinosinusitis with nasal polyps (EosCRSwNPs), but the regulation mechanism of dendritic cells (DCs) maturation is still unclear. We identified nasal mucosa of 20 patients with EosCRSwNP, 16 non-EosCRSwNP patients, and inferior turbinate of 14 patients with nasal septum deviation after surgery. The expression of cyclin-dependent kinase 5 (CDK5) and programmed cell death 1 ligand 1 (PD-L1) were detected by immunofluorescent, real-time quantitative PCR, and Western blot in EosCRSwNP. The level of dendritic cell maturation was detected by flow cytometry and immunofluorescence staining after CDK5 expression interference with small interfering RNA (siRNA). The expression of CDK5 and PD-L1 in EosCRSwNP nasal mucosal tissue was significantly higher than that of non-EosCRSwNP and inferior turbinate nasal mucosa tissue, and there was a positive correlation between them. Immunofluorescence staining showed that CDK5 and PD-L1 were co-localized in dendritic cells. Synergistic stimulation of dendritic cells with LPS and TNF-α promotes the maturation of dendritic cells and increases the expression of CDK5 and PD-L1. However, blocking the expression of CDK5 in dendritic cells with siRNAs leads to a blockage of cell maturation. CDK5 can regulate the expression of PD-L1, and its presence is critical for the maturation of dendritic cells. CDK5 may play an important role in the pathogenesis of CRSwNP disease.

Modulating Protein Phosphatase 2A Rescues Disease Phenotype in Neurodegenerative Tauopathies.

Alzheimer's disease (AD) is the leading cause of dementia worldwide accounting for around 70% of all cases. There is currently no treatment for AD beyond symptom management and attempts at developing disease-modifying therapies have yielded very little. These strategies have traditionally targeted the peptide Aß, which is thought to drive pathology. However, the lack of clinical translation of these Aß-centric strategies underscores the need for diverse treatment strategies targeting other aspects of the disease. Metal dyshomeostasis is a common feature of several neurodegenerative diseases such as AD, Parkinson's disease, and frontotemporal dementia, and manipulation of metal homeostasis has been explored as a potential therapeutic avenue for these diseases. The copper ionophore glyoxalbis-[N4-methylthiosemicarbazonato]Cu(II) (CuII(gtsm)) has previously been shown to improve the cognitive deficits seen in an AD animal model; however, the molecular mechanism remained unclear. Here we report that the treatment of two animal tauopathy models (APP/PS1 and rTg4510) with CuII(gtsm) recovers the cognitive deficits seen in both neurodegenerative models. In both models, markers of tau pathology were significantly reduced with CuII(gtsm) treatment, and in the APP/PS1 model, the levels of Aß remained unchanged. Analysis of tau kinases (GSK3ß and CDK5) revealed no drug induced changes; however, both models exhibited a significant increase in the levels of the structural subunit of the tau phosphatase, PP2A. These findings suggest that targeting the tau phosphatase PP2A has therapeutic potential for preventing memory impairments and reducing the tau pathology seen in AD and other tauopathies.

Persistent histone modifications at the BDNF and Cdk-5 promoters following extinction of nicotine-seeking in rats.

Drugs of addiction lead to a wide range of epigenetic changes at the promoter regions of genes directly implicated in learning and memory processes. We have previously shown that the histone deactylase inhibitor, sodium butyrate (NaB), accelerates the extinction of nicotine-seeking and provides resistance to relapse. Here, we explore the potential molecular mechanisms underlying this effect. Rats received intravenous nicotine or saline self-administration, followed by 6 days of extinction training, with each extinction session followed immediately by treatment with NaB or vehicle. On the last day of extinction, rats were killed and the medial ventral prefrontal cortex retained for chromatin immunoprecipitation and quantitative polymerase chain reaction (qPCR). A history of nicotine exposure significantly decreased H3K14 acetylation at the brain-derived neurotrophic factor (BDNF) exon IV promoter, and this effect was abolished with NaB treatment. In contrast, nicotine self-administration alone, resulted in a significant decrease in histone methylation at the H3K27me3 and H3K9me2 marks in the promoter regions of BDNF exon IV and cyclin-dependent kinase 5 (Cdk-5). Quantitative PCR-identified changes in several genes associated with NaB treatment that were independent of nicotine exposure; however, an interaction of nicotine history and NaB treatment was detected only in the expression of BDNF IV and BDNF IX. Together these results suggest that nicotine self-administration leads to a number of epigenetic changes at both the BDNF and Cdk-5 promoters, and that these changes may contribute to the enhanced extinction of nicotine-seeking by NaB.

Retinoic acid increases glucocorticoid receptor phosphorylation via cyclin-dependent kinase 5.

Glucocorticoid receptor (GR) function is modulated by phosphorylation. As retinoic acid (RA) can activate some cytoplasmic kinases able to phosphorylate GR, we investigated whether RA could modulate GR phosphorylation in neuronal cells in a context of long-term glucocorticoid exposure. A 4-day treatment of dexamethasone (Dex) plus RA, showed that RA potentiated the (Dex)-induced phosphorylation on GR Serine 220 (pSer220GR) in the nucleus of a hippocampal HT22 cell line. This treatment increased the cytoplasmic ratio of p35/p25 proteins, which are major CDK5 cofactors. Roscovitine, a pharmacological CDK5 inhibitor, or a siRNA against CDK5 prevented RA potentiation of GR phosphorylation. Furthermore, roscovitine counter-acted the effect of RA on GR sensitive target proteins such as BDNF or tissue-transglutaminase. These data help understanding the interaction between RA- and glucocorticoid-signalling pathways, both of which have strong influences on the adult brain.

Dissociative role for dorsal hippocampus in mediating heroin self-administration and relapse through CDK5 and RhoB signaling revealed by proteomic analysis.

Addiction is characterized by drug craving, compulsive drug taking and relapse, which is attributed to aberrant neuroadaptation in brain regions implicated in drug addiction, induced by changes in gene and protein expression in these regions after chronic drug exposure. Accumulating evidence suggests that the dorsal hippocampus (DH) plays an important role in mediating drug-seeking and drug-taking behavior and relapse. However, the molecular mechanisms underlying these effects of the DH are unclear. In the present study, we employed a label-free quantitative proteomic approach to analyze the proteins altered in the DH of heroin self-administering rats. A total of 4015 proteins were quantified with high confidence, and 361 proteins showed significant differences compared with the saline control group. Among them, cyclin-dependent kinase 5 (CDK5) and ras homolog family member B (RhoB) were up-regulated in rats with a history of extended access to heroin. Functionally, inhibition of CDK5 in the DH enhanced heroin self-administration, indicating that CDK5 signaling in the DH acts as a homeostatic compensatory mechanism to limit heroin-taking behavior, whereas blockade of the Rho-Rho kinase (ROCK) pathway attenuated context-induced heroin relapse, indicating that RhoB signaling in the DH is required for the retrieval (recall) of addiction memory. Our findings suggest that manipulation of CDK5 signaling in the DH may be essential in determining vulnerability to opiate taking, whereas manipulation of RhoB signaling in the DH may be essential in determining vulnerability to relapse. Overall, the present study suggests that the DH can exert dissociative effects on heroin addiction through CDK5 and RhoB signaling.

Sex differences in behavioral and PKA cascade responses to repeated cocaine administration.

Previous studies have shown sex different patterns in behavioral responses to cocaine. Here, we used between-subject experiment design to study whether sex differences exist in the development of behavioral sensitization and tolerance to repeated cocaine, as well as the role of protein kinase A (PKA) signaling cascade in this process. Ambulatory and rearing responses were recorded in male and female rats after 1 to 14 days of administration of saline or cocaine (15 mg/kg; ip). Correspondent PKA-associated signaling in the nucleus accumbens (NAc) and caudate-putamen (CPu) was measured at each time point. Our results showed that females exhibited higher cocaine-induced behavioral responses and developed behavioral sensitization and tolerance faster than males. Whereas females developed behavioral sensitization to cocaine after 2 days and tolerance after 14 days, male rats developed sensitization after 5 days. In addition, cocaine induced a sexual dimorphic pattern in the progression of neuronal adaptations on the PKA cascade signaling in region (NAc vs. CPu) and time (days of cocaine administration)-dependent manners. In general, more PKA signaling cascade changes were found in the NAc of males on day 5 and in the CPu of females with repeated cocaine injection. In addition, in females, behavioral activities positively correlated with FosB levels in the NAc and CPu and negatively correlated with Cdk5 and p35 in the CPu, while no correlation was observed in males. Our studies suggest that repeated cocaine administration induced different patterns of behavioral and molecular responses in the PKA cascade in male and female rats.

Targeting cyclin dependent kinase 5 in hepatocellular carcinoma--A novel therapeutic approach.

BACKGROUND & AIMS: For a long time cyclin dependent kinase 5 (Cdk5) was thought to be exclusively important in neuronal cells. However, increasing evidence recently suggests a function of Cdk5 in cancer progression. In this study, we examined the role of Cdk5 and its therapeutic accessibility in hepatocellular carcinoma (HCC), a highly chemoresistant cancer with poor prognosis and paramount clinical importance in order to develop novel targeted therapies for systemic treatment. METHODS: Expression and activity of Cdk5 was analyzed in a human HCC tissue microarray, human patient samples and HCC cell lines. To characterize Cdk5 functions and signaling pathways in HCC, we applied genetic downregulation and pharmacologic inhibition in various approaches including cell based assays and mouse xenograft models. RESULTS: Expression and activity of Cdk5 was increased in human HCC tissues as compared to normal liver tissues. Functional ablation of Cdk5 significantly decreased HCC cell proliferation and clonogenic survival. Moreover, genetic and pharmacological inhibition of Cdk5 showed in vivo efficacy in HCC xenograft mouse models. Investigating the mechanisms behind these functional effects revealed that Cdk5 is most active in the nucleus of cells in G2/M phase. Cdk5 regulates DNA damage response by phosphorylating ataxia telangiectasia mutated (ATM) kinase and thereby influencing its downstream cascade. Consequently, combination of Cdk5 inhibition with DNA-damage-inducing chemotherapeutics synergistically inhibited HCC tumor progression in vitro and in vivo. CONCLUSIONS: In summary, we introduce Cdk5 as a novel drugable target for HCC treatment and suggest the combination of Cdk5 inhibition and DNA damaging agents as a novel therapeutic approach.

Neuroprotective effects of the anti-cancer drug sunitinib in models of HIV neurotoxicity suggests potential for the treatment of neurodegenerative disorders.

BACKGROUND AND PURPOSE: Anti-retrovirals have improved and extended the life expectancy of patients with HIV. However, as this population ages, the prevalence of cognitive changes is increasing. Aberrant activation of kinases, such as receptor tyrosine kinases (RTKs) and cyclin-dependent kinase 5 (CDK5), play a role in the mechanisms of HIV neurotoxicity. Inhibitors of CDK5, such as roscovitine, have neuroprotective effects; however, CNS penetration is low. Interestingly, tyrosine kinase inhibitors (TKIs) display some CDK inhibitory activity and ability to cross the blood-brain barrier. EXPERIMENTAL APPROACH: We screened a small group of known TKIs for a candidate with additional CDK5 inhibitory activity and tested the efficacy of the candidate in in vitro and in vivo models of HIV-gp120 neurotoxicity. KEY RESULTS: Among 12 different compounds, sunitinib inhibited CDK5 with an IC50 of 4.2 µM. In silico analysis revealed that, similarly to roscovitine, sunitinib fitted 6 of 10 features of the CDK5 pharmacophore. In a cell-based model, sunitinib reduced CDK5 phosphorylation (pCDK5), calpain-dependent p35/p25 conversion and protected neuronal cells from the toxic effects of gp120. In glial fibrillary acidic protein-gp120 transgenic (tg) mice, sunitinib reduced levels of pCDK5, p35/p25 and phosphorylated tau protein, along with amelioration of the neurodegenerative pathology. CONCLUSIONS AND IMPLICATIONS: Compounds such as sunitinib with dual kinase inhibitory activity could ameliorate the cognitive impairment associated with chronic HIV infection of the CNS. Moreover, repositioning existing low MW compounds holds promise for the treatment of patients with neurodegenerative disorders.

The expression of intermediate filament protein nestin and its association with cyclin-dependent kinase 5 in the glomeruli of rats with diabetic nephropathy.

BACKGROUND: Podocyte injury plays a crucial role in the development of diabetic nephropathy (DN), but its underlying mechanism remains poorly understood. Emerging evidences suggest that the cytoskeleton disruption is related to podocyte injury. The aim of this study was to investigate whether nestin, a cytoskeleton-associated intermediate filament protein, is involved in the development of DN. METHODS: Rat diabetes was induced by intraperitoneal injection of streptozotocin. The renal histological changes were investigated by light microscopy and transmission electron microscopy. The location of nestin and vimentin in renal tissues was observed by immunohistochemistry. The protein or messenger RNA levels of nestin and cyclin-dependent kinase 5 (Cdk5) were detected by Western blot and real-time polymerase chain reaction. The relationship between nestin and vimentin was detected by co-immunoprecipitation. RESULTS: Compared with controls, diabetic rats showed significant characteristics of renal damage. The expression of nestin and vimentin in the glomeruli was increased at the early stage of diabetes, which then gradually decreased. Co-immunoprecipitation assays demonstrated that nestin disassembled with vimentin in diabetic rats. The expression of Cdk5 was increased in a time-dependent manner in diabetic rats. The degree of albuminuria in diabetic rats was negatively correlated with nestin and positively correlated with Cdk5. Roscovitine, a Cdk5 inhibitor, reduced the degradation of nestin. Moreover, podocyte injuries were significantly ameliorated by treatment with roscovitine. CONCLUSIONS: The intermediate filament protein nestin is associated with development of DN. Blockage of Cdk5 increases the level of nestin and attenuates renal damage, which would provide a useful target for DN therapy.

Tau hyperphosphorylation and axonal damage induced by N,N-diethyldithiocarbamate (DEDTC) treatment along late postnatal development is followed by a rescue during adulthood.

Axonal degeneration has been described as the pathological hallmark of peripheral neuropathies induced by DEDTC. In addition, axonal damage has also been observed in the brain of mice treated daily with DEDTC along postnatal development, though with this experimental model there was observed to be axonal recovery after treatment, during the adulthood. To focus on this axonal dynamic activity, damage-recovery, a key axonal protein, the microtubule associated protein tau, was analyzed in this DEDTC model. Tau is a phosphoprotein and its dynamic site-specific phosphorylation is essential for its proper function; in fact, high levels are correlated with cell dysfunction. Furthermore, the levels of tau phosphorylation are associated with dynamic microtubules during periods of high plasticity. Thus, phosphorylated tau at two sites of phosphorylation, Ser(199) and Ser(396), were evaluated during the second week of postnatal development and throughout adulthood. The results obtained by Western blot made it evident that the levels of p-tau Ser(199) and p-tau Ser(396) were higher in treated mice than in controls. Interestingly, by immunohistochemistry there was shown to be an increase in p-tau-immunolabeling in neuronal soma together with axonal tract alterations in treated animals with respect to controls, and the analyses of GSK3 beta and cdk5 revealed an increase in its activity in DEDTC-treated animals. Nevertheless, in the adult a general decline in p-tau was observed together with a rescue of axonal tract. All these data support the idea that the axonal damage induced by DEDTC treatment along postnatal development is followed by an axonal rescue during adulthood.

CD40 ligation mediates plaque-associated tau phosphorylation in beta-amyloid overproducing mice.

Neuritic dystrophy with amyloid burden and neurofibrillary tangles are pathological hallmarks of Alzheimer's disease. Genetic disruption of CD40 or CD40L alleviates amyloid burden, astrocytosis, and microgliosis in transgenic animal models of Alzheimer's disease. It has been reported that phosphorylated tau-positive dystrophic neurites are observed in transgenic mice over-expressing human mutant beta-amyloid precursor protein (Tg2576). Here, we studied the pattern of phosphorylated tau (labeled with AT8, CP13, PG5, and PHF1 antibodies) and plaques using immunohistochemical techniques. Phosphorylated tau-positive dystrophic neurites were exclusively associated with Congo red-positive plaques as previously reported. Further, we show that CD40L or CD40 deficiency reduces the mean ratio of dystrophic neurite area to congophilic plaque area and the level of expression of cdk5 and p35/p25 in mice. In addition, we show that in a human neuroblastoma cell line treated with CD40L, cdk5 and p35/p25 are increased. Together, our data suggest that CD40-CD40L interaction has an effect on tau phosphorylation independent of beta-amyloid pathology, and that this effect may occur through a decrease of cdk5 and p35/p25.

Lithium treatment decreases activities of tau kinases in a murine model of senescence.

Lithium modulates glycogen synthase kinase 3beta (GSK-3beta), a kinase involved in Alzheimer disease-related tau pathology. To investigate mechanisms of aging and the potential therapy of lithium in neurodegenerative disease, we treated senescence-accelerated mouse (SAM)P8 mice, a murine model of senescence, and mice of the control SAMR1 strain with lithium. The treatment reduced hippocampal caspase 3 and calpain activation, indicating that it provides neuroprotection. Lithium also reduced both the levels and activity of GSK-3beta and the activity of cyclin-dependent kinase 5 and reduced hyperphosphorylation of 3 different phosphoepitopes of tau: Ser199, Ser212, and Ser396. In lithium-treated primary cultures of SAMP8 and SAMR1 cerebellar neurons, there was a marked reduction in protease activity mediated by calpain and caspase 3. Both lithium and SB415286, a specific inhibitor of GSK-3beta, reduced apoptosis in vitro. Taken together, these in vivo and in vitro findings of lithium-mediated reductions in GSK-3beta and cyclin-dependent kinase 5 activities, tau phosphorylation, apoptotic activity, and cell death provide a strong rationale for the use of lithium as a potential treatment in neurodegenerative diseases.

Behavioral expression of cocaine sensitization in rats is accompanied by a distinct pattern of modifications in the PKA/DARPP-32 signaling pathway.

Repeated cocaine administration induces behavioral sensitization and modifications in the phosphorylation pattern of dopamine and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32), characterized by a tonic increase in the Thr75 phosphorylated form, and a decrease in the Thr34 phosphorylated form. This study further investigated the correlations between cocaine sensitization and modifications in the DARPP-32 phosphorylation pattern, cAMP-dependent protein kinase (PKA) activity, and mGluR5 tone in the medial prefrontal cortex and nucleus accumbens. Behavioral sensitization and modifications in these neurochemical markers followed a similar temporal pattern. Moreover, in sensitized rats acute cocaine administration modified phosphorylation levels of Thr75- and Thr34-DARPP-32, GluR1, and NR1 subunits in the nucleus accumbens only at a dose double the efficacious dose in control rats. These results suggest that the high levels of phospho-Thr75 DARPP-32 maintain PKA in a prevalent inhibited state. Furthermore, in sensitized rats the acute administration of 6-methyl-2-(phenylethynyl)-pyridine, a mGluR5 antagonist, reinstated the phosphorylation levels of Thr75- and Thr34-DARPP-32, GluR1, and NR1 to control values, and a subsequent cocaine challenge did not elicit a sensitized response. These data suggest that a tonic increase in mGluR5 transmission in cocaine-sensitized rats sustains both the increase in phospho-Thr75 DARPP-32 levels and the expression of behavioral sensitization.

Cyclin-dependent kinase 5 differentially regulates the transcriptional activity of the glucocorticoid receptor through phosphorylation: clinical implications for the nervous system response to glucocorticoids and stress.

Glucocorticoids, major end effectors of the stress response, play an essential role in the homeostasis of the central nervous system and influence diverse functions of neuronal cells. We found that cyclin-dependent kinase 5 (CDK5), which plays important roles in the morphogenesis and functions of the nervous system and whose aberrant activation is associated with development of neurodegenerative disorders, interacted with the ligand-binding domain of the glucocorticoid receptor (GR) through its activator p35 or its active proteolytic fragment p25. CDK5 phosphorylated GR at multiple serines, including Ser203 and Ser211 of its N-terminal domain, and suppressed the transcriptional activity of this receptor on glucocorticoid-responsive promoters by attenuating attraction of transcriptional cofactors to DNA. In microarray analyses using rat cortical neuronal cells, the CDK5 inhibitor roscovitine differentially regulated the transcriptional activity of the GR on more than 90% of the endogenous glucocorticoid-responsive genes tested. Thus, CDK5 exerts some of its biological activities in neuronal cells through the GR, dynamically modulating GR transcriptional activity in a target promoter-dependent fashion.

Increased accumbens Cdk5 expression in rats after short-access to self-administered cocaine, but not after long-access sessions.

Upregulation of cyclin-dependent kinase 5 (Cdk5) after chronic cocaine administration has led to speculation that Cdk5 plays an important role in drug addiction. However, as Cdk5 involvement is implicated in a variety of neural events, including neuronal development, synaptic plasticity and learning, a specific role in drug abuse is yet to be determined. The present study utilized cocaine self-administration and food-reinforced operant procedures to assess possible relationships between cocaine intake, food-reinforced operant responding, behavioral activity, and Cdk5 levels in the nucleus accumbens (NAcc), ventral tegmental area (VTA), and prefrontal cortex (PFC) in rats. In Experiment 1, animals undergoing daily cocaine self-administration (1-h/30 days) or food-reinforced operant sessions (20-min/30 days) showed significant between-group differences in operant responding and behavioral activity, but no significant differences in NAcc, VTA or PFC Cdk5 levels compared to a Handled Control group. In Experiment 2, animals that had self-administered cocaine in 10 daily 1-h sessions (Short-Access Cocaine) showed significantly greater NAcc Cdk5 expression compared to an Unhandled Control group, and no evidence of cocaine-induced behavioral sensitization. Animals given 4-h daily access to cocaine over the same number of sessions (Long-Access Cocaine) showed significantly enhanced cocaine-reinforced responding and locomotor activation by the end of the sessions, but no significant differences in Cdk5 expression compared to Control animals. These findings suggest that overexpression of Cdk5 may be a transient adaptation to cocaine experience that subsides with increased cocaine exposure and does not correspond with measures of cocaine-induced behavioral sensitization.

Roscovitine regulates invasive breast cancer cell (MDA-MB231) proliferation and survival through cell cycle regulatory protein cdk5.

Roscovitine, a purine analogue, has been considered for the treatment of cancer. Anti-cancer therapeutic efficacy is being evaluated in clinical trials. However, the mechanisms remain unclear. In the present study, cyclic-dependent kinase 5 (cdk5) proved to be a molecular target for roscovitine-triggered apoptosis for highly invasive breast cancer cell death. Because our previous studies have shown a potential role of cdk5 in endothelial cell proliferation/apoptosis [Sharma, M.R., Tuszynski, G.P., Sharma, M.C. (2004). Angiostatin-induced inhibition of endothelial cell proliferation/apoptosis is associated with the down-regulation of cell cycle regulatory protein cdk5. J. Cell Biochem. 91, 398-409], here we not only demonstrate first that Cdk5, p35, and p25 proteins were all expressed in invasive breast cancer cells MDA-MB231 but also showed that cdk5 expression regulates MDA-MB231 cell proliferation. In addition, potent mitogen bFGF up-regulates cdk5 expression. Roscovitine specifically inhibits cdk5 expression/activity in a dose-dependent manner with concomitant inhibition of MDA-MB231 cell proliferation and induction of apoptosis. By contrast, the roscovitine analog olomoucine, a specific inhibitor of cdk4, failed to affect MDA-MB231 cell proliferation and apoptosis which implies the specific involvement of cdk5 in roscovitine-triggered cell death/proliferation. Additionally, roscovitine-mediated inhibition of proliferation is irreversible. These data suggest that cdk5 may have a significant role in the regulation of breast cancer cell proliferation and apoptosis and extend beyond its role in neurogenesis. These results suggest that Cdk5 is a novel player in roscovitine-triggered breast cancer cell apoptosis and inhibition of proliferation, therefore, may be a potential therapeutic target.