Cyclooxygenase-2 (COX2) is a primary inflammatory mediator that converts arachidonic acid into precursors of vasoactive prostaglandins, producing reactive oxygen species in the process. Under normal conditions COX2 is not detectable, except at low abundance in the brain. This study demonstrates a distinctive pattern of COX2 increases in the brain over time following traumatic brain injury (TBI). Quantitative lysate ribonuclease protection assays indicate acute and sustained increases in COX2 mRNA in two rat models of TBI. In the lateral fluid percussion model, COX2 mRNA is significantly elevated (>twofold, p < 0.05, Dunnett) at 1 day postinjury in the injured cortex and bilaterally in the hippocampus, compared to sham-injured controls. In the lateral cortical impact model (LCI), COX2 mRNA peaks around 6 h postinjury in the ipsilateral cerebral cortex (fivefold induction, p < 0.05, Dunnett) and in the ipsilateral and contralateral hippocampus (two-and sixfold induction, respectively, p < 0.05, Dunnett). Increases are sustained out to 3 days postinjury in the injured cortex in both models. Further analyses use the LCI model to evaluate COX2 induction. Immunoblot analyses confirm increased levels of COX2 protein in the cortex and hippocampus. Profound increases in COX2 protein are observed in the cortex at 1-3 days, that return to sham levels by 7 days postinjury (p < 0.05, Dunnett). The cellular pattern of COX2 induction following TBI has been characterized using immunohistochemistry. COX2-immunoreactivity (-ir) rises acutely (cell numbers and intensity) and remains elevated for several days following TBI. Increases in COX2-ir colocalize with neurons (MAP2-ir) and glia (GFAP-ir). Increases in COX2-ir are observed in cerebral cortex and hippocampus, ipsilateral and contralateral to injury as early as 2 h postinjury. Neurons in the ipsilateral parietal, perirhinal and piriform cortex become intensely COX2-ir from 2 h to at least 3 days postinjury. In agreement with the mRNA and immunoblot results, COX2-ir appears greatest in the contralateral hippocampus. Hippocampal COX2-ir progresses from the pyramidal cell layer of the CA1 and CA2 region at 2 h, to the CA3 pyramidal cells and dentate polymorphic and granule cell layers by 24 h postinjury. These increases are distinct from those observed following inflammatory challenge, and correspond to brain areas previously identified with the neurological and cognitive deficits associated with TBI. While COX2 induction following TBI may result in selective beneficial responses, chronic COX2 production may contribute to free radical mediated cellular damage, vascular dysfunction, and alterations in cellular metabolism. These may cause secondary injuries to the brain that promote neuropathology and worsen behavioral outcome.
CDK9 is a CDC2-related kinase and the catalytic subunit of the positive-transcription elongation factor b and the Tat-activating kinase. It has recently been reported that CDK9 is a short-lived protein whose levels are regulated during the cell cycle by the SCF SKP2 ubiquitin ligase complex (R. E. Kiernan et al., Mol. Cell. Biol. 21:7956-7970, 2001). The results presented here are in contrast to those observations. CDK9 protein levels remained unchanged in human cells entering and progressing through the cell cycle from G 0 , despite dramatic changes in SKP2 expression. CDK9 levels also remained unchanged in cells exiting from mitosis and progressing through the next cell cycle. Similarly, the levels of CDK9 protein did not change as cells exited the cell cycle and differentiated along various lineages. In keeping with these observations, the kinase activity associated with CDK9 was found to not be regulated during the cell cycle. We have also found that endogenous CDK9 is a very stable protein with a half-life (t 1/2 ) of 4 to 7 h, depending on the cell type. In contrast, when CDK9 is overexpressed, it is not stabilized and is rapidly degraded, with a t 1/2 of less than 1 h, depending on the level of expression. Treatment of cells with proteasome inhibitors blocked the degradation of short-lived proteins, such as p27, but did not affect the expression of endogenous CDK9. Ectopic overexpression of SKP2 led to reduction of p27 protein levels but had no effect on the expression of endogenous CDK9. Finally, downregulation of endogenous SKP2 gene expression by interfering RNA had no effect on CDK9 protein levels, whereas p27 protein levels increased dramatically. Therefore, the SCF SKP2 ubiquitin ligase does not regulate CDK9 expression in a cell cycle-dependent manner.CDK9 is the catalytic subunit of the positive-transcription elongation factor b (P-TEFb) (30). CDK9 was first identified as a CDC2-related kinase named PITALRE, which associated with a number of cellular polypeptides and exhibited a kinase activity that was not regulated during the cell cycle (14). CDK9 associates with either cyclin T1, T2a, T2b, or K to form active P-TEFb complexes, which activate transcriptional elongation by phosphorylating the C-terminal domain (CTD) of RNA polymerase II (RNAPII) as well as negative-transcription elongation factors (reviewed in reference 26). CDK9 is tethered to promoters by certain transcription factors, such as NF-B, the androgen receptor, and c-myc, resulting in stimulation of transcription (2, 8, 18). CDK9/cyclin T1 complexes are also the target of the human immunodeficiency virus transcriptional activator Tat, which recruits this complex to the transactivation response structure in the nascent human immunodeficiency virus RNA and positions the kinase complex to phosphorylate the CTD of the RNAPII, and other transcription factors, resulting in stimulation of transcriptional elongation that would otherwise be interrupted (26).CDK9 has also been found to associate with components of a chaperone pathway, includin...
Stimulation of primary human T lymphocytes results in up-regulation of cyclin T1 expression, which correlates with phosphorylation of the C-terminal domain of RNA polymerase II (RNAP II). Up-regulation of cyclin T1 and concomitant stabilization of cyclin-dependent kinase 9 (CDK9) may facilitate productive replication of HIV in activated T cells. We report that treatment of PBLs with two mitogens, PHA and PMA, results in accumulation of cyclin T1 via distinct mechanisms. PHA induces accumulation of cyclin T1 mRNA and protein, which results from cyclin T1 mRNA stabilization, without significant change in cyclin T1 promoter activity. Cyclin T1 mRNA stabilization requires the activation of both calcineurin and JNK because inhibition of either precludes cyclin T1 accumulation. In contrast, PMA induces cyclin T1 protein up-regulation by stabilizing cyclin T1 protein, apparently independently of the proteasome and without accumulation of cyclin T1 mRNA. This process is dependent on Ca2+-independent protein kinase C activity but does not require ERK1/2 activation. We also found that PHA and anti-CD3 Abs induce the expression of both the cyclin/CDK complexes involved in RNAP II C-terminal domain phosphorylation and the G1-S cyclins controlling cell cycle progression. In contrast, PMA alone is a poor inducer of the expression of G1-S cyclins but often as potent as PHA in inducing RNAP II cyclin/CDK complexes. These findings suggest coordination in the expression and activation of RNAP II kinases by pathways that independently stimulate gene expression but are insufficient to induce S phase entry in primary T cells.
HIV-1 transcription is essential for the virus replication cycle. HIV-1 Tat is a viral transactivator that strongly stimulates the processivity of RNA polymerase II (RNAPII) via recruitment of the cyclin T1/CDK9 positive transcription elongation factor, which phosphorylates the C-terminal domain (CTD) of RNAPII. Consistently, HIV-1 replication in transformed cells is very sensitive to direct CDK9 inhibition. Thus, CDK9 could be a potential target for anti-HIV-1 therapy. A clearer understanding of the requirements for CDK9 activity in primary human T cells is needed to assess whether the CDK9-dependent step in HIV-1 transcription can be targeted clinically. We have investigated the effects of limiting CDK9 activity with recombinant lentiviruses expressing a dominant negative form of CDK9 (HA-dnCDK9) in Peripheral Blood Lymphocytes (PBLs) and other cells. Our results show that direct inhibition of CDK9 potently inhibits HIV-1 replication in single-round infection assays with little to undetectable effects on RNAPII transcription, RNA synthesis, proliferation and viability. In PBLs purified from multiple donors, direct inhibition of CDK9 activity blocks HIV-1 replication/transcription but does not prevent T cell activation, as determined via measurement of cell surface and cell cycle entry and progression markers, and DNA synthesis. We have also compared the effects of HA-dnCDK9 to flavopiridol (FVP), a general CDK inhibitor that potently inhibits CDK9. In contrast to HA-dnCDK9, FVP interferes with key cellular processes at concentrations that inhibit HIV-1 replication with potency similar to HA-dnCDK9. In particular, FVP inhibits several T cell activation markers and DNA synthesis in primary PBLs at the minimal concentrations required to inhibit HIV-1 replication. Our results imply that small pharmacological compounds targeting CDK9 with enhanced selectivity could be developed into effective anti-HIV-1 therapeutic drugs.
This review primarily focuses on the mechanisms that modulate CDK9 activity and its recruitment to cellular genes, where it phosphorylates the C-terminal domain of RNA polymerase II (RNAPII) as well as negative elongation factors. CDK9 associates with each of four cyclins (T1, T2a, T2b and K), forming distinct positive transcription elongation factors (P-TEFb). Research done during the past decade has demonstrated a role for P-TEFb in stimulating elongation of otherwise paused RNAPII transcripts. Recent work suggests that P-TEFb also positively modulates other steps during transcription. In addition, "abnormal" CDK9 function is associated with certain diseases. Specifically, the activity of the cyclin T1/CDK9 complex is essential for HIV-1 replication and CDK9 upregulation is associated with cardiac hypertrophy. Thus, the role of CDK9 in these processes, and the possibility of therapeutically targeting CDK9, will also be briefly discussed.
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