In the present study, we firstly revealed that CDK9 was upregulated in isolated pulmonary arterial tissues from MCT-induced PH rats in vivo and in hypoxic cultured HPASMCs in vitro. This upregulation of CDK9 was associated with increased CDK9-mediated phosphorylation of the C-terminal domain (CTD) of RNA pol II at serine-2. Secondly, several downstream prosurvival and antiapoptotic proteins (c-Myc, Mcl-1 and survivin) were also upregulated at both the mRNA and protein levels. These molecular changes in pulmonary arterial tissues coincident with pathogenic enhancement of pulmonary vasculature remodeling and elevated pulmonary arterial pressure. Furthermore, flavopiridol significantly alleviated pulmonary artery remodeling and reversed the progression of pulmonary hypertension through the inhibition of CDK9-meditated transcription elongation. Taken together, these findings elucidated the potential role and underlying mechanism of CDK9 in the pathogenesis of pulmonary vasculature remodeling under PH and flavopiridol can partially reverse pathogenic vasculature remodeling in PH rats by targeting CDK9.
Growing evidence indicates that pulmonary hypertension shares several similar pathogenic characteristics with the cancers [6, 7], with one of the most important similarities being that both cancer cells and PH-associated vascular cells, especially PASMCs, exhibiting a proproliferative and antiapoptotic phenotype [3, 6]. In our present study, the level of a biomarker of cell proliferation (PCNA) in pulmonary arteries from MCT-induced PH rats were increased, and a significant hypertrophic media layer harboring proliferative PASMCs was observed in pulmonary small arteries. This overproliferation phenotype of vascular cells, especially PASMCs, will lead to an increasing thickness of the pulmonary artery media wall, consequently inducing PH and right ventricle failure. Moreover, HPASMCs overproliferation and resistance to apoptosis were also induced under hypoxic (3%) conditions, similar to that observed in cancer cells exposed to a hypoxic environment [32]. Interestingly, this abnormal overproliferative and antiapoptotic phenotype of PAMSCs can be reversed by treatment with the antitumor agent flavopiridol in both in vivo PH model and in vitro HPASMCs, suggesting that reversing these cancer-like phenotypes may be a promising therapeutic strategy to combat PH.
CDK9 has been extensively investigated as a target for cancer therapy, and CDK9 upregulation has been shown to play a crucial role in the proproliferative and antiapoptotic phenotype of cancer cells [18, 20]. CDK9 overexpression has been reported in osteosarcoma, melanoma, gastric cancer, etc [16, 17, 33]. CDK9 inhibition blocks transcriptional elongation, thereby suppressing the expression of antiapoptotic proteins, such as MCL-1, BIRC513. CDK9 inhibitors such as flavopiridol, SNS-032, or roscovitine have proven efficient through the downregulation of anti-apoptotic genes XIAP and MCL-1[16, 26, 34]. Although several signal-responsive genes that regulate proliferation and apoptosis, such as proto-oncogenes proteins (c-Myc), antiapoptotic proteins (Mcl-1 and survivin), have been attributed to CDK9 related abnormal transcriptional elongation, and elevated CDK9 expression was also reported in cancer cells [12, 18, 20], its potential roles in PH pathogenesis remain largely unknown.
Flavopiridol is a broadly specific CDK inhibitor with a distinct preference for CDK9. The Ki values of flavopiridol for CDK9/CycT (3 nM) was previously shown to be approximately 10-fold lower than those for other CDKs (40-70 nM) [35]. CDK9 inhibition by flavopiridol can remarkably attenuate tumor growth in vitro and in vivo [26, 27]. As previously observed in cancer cells, CDK9 upregulation was also detected in both pulmonary arterial tissues from MCT-induced PH rats and hypoxia-treated HPASMCs. Additionally, CDK9 inhibition by flavopiridol attenuated the overproliferation and promoted the apoptosis of PASMCs, thereby alleviating pulmonary vasculature remodeling and reversing the progression of pulmonary hypertension in rats, which is like its antitumor effects. Taken together, these results show that CDK9 upregulation also contributes to the proproliferative and anti-apoptotic phenotype of PH PASMCs, which is like that observed in cancer cells.
As a catalytic subunit of P-TEFb, CDK9 activation can promote transcriptional elongation by phosphorylating RNA polymerase II CTD. In the present study, consistent with its potent inhibition of CDK9 kinase activity observed in cancer cells [26], flavopiridol also significantly reduced the phosphorylation on RNA polymerase II CTD at Ser-2 in isolated PH pulmonary arterial tissues, indicating a potential role of flavopiridol in reversing pulmonary vessel remolding. Furthermore, the overexpression of several short-lived prosurvival and antiapoptotic proteins, such as c-Myc, Mcl-1 and survivin has been reported to contribute to overproliferation and resistance to apoptosis in cancer cells [19, 20], and these proteins were preferentially depleted by the inhibition of CDK9-mediated transcriptional elongation [13]. In agreement with the results of other studies, both mRNA and protein levels of c-Myc, Mcl-1 and survivin were also significantly increased in our isolated PH pulmonary arterial tissues, which could be remarkably decreased by flavopiridol treatment. Therefore, our results suggested that the antiproliferative and proapoptotic effects of flavopiridol on pulmonary arteries and PASMCs are mediated by the inhibition of CDK9-mediated transcriptional elongation and these expressions of these downstream proteins.
In addition to the changes of CDK9 expression or activity,CDK9/P-TEFb related regulators may also be involved in CDK9-related disease (e.g. cancers) [28, 30, 36]. As previously reported, LARP7, HEXIM1 or HEXIM2 and MePCE, bind to 7SK small nuclear RNA (snRNA) to form the 7SK small nuclear ribonucleoprotein (7SK snRNP) complex, which plays a role in inhibiting CDK9/P-TEFb activity [29, 30]. LARP7 was previously reported to suppress CDK9/P-TEFb activity, and LARP7 knockdown or inhibition can cause an increase in CDK9 activity [30]. HEXIM1/2 can inhibit CDK9 kinase activity in a 7SK snRNA -dependent manner [29]. MePCE was reported to stabilize 7SK snRNA to facilitate the inhibition of CDK9/P-TEFb activity [37]. In agreement with these findings, in our present study, these components of the 7SK snRNP complex [LARP7, HEXIM1 (not HEXIM2) and MePCE] were significantly decreased in isolated PH pulmonary arteries, suggesting that these CDK9-related negative regulators may also be involved in the pathology of pulmonary hypertension. Additionally, previous studies have reported that overexpression of these CDK9-related negative regulators (e.g., LARP7) decreases CDK9/P-TEFb kinase activity to reverse the progression of CDK9-related diseases [30, 36], thus, the underlying mechanisms of these CDK9-related negative regulators in pulmonary hypertension requires further investigation.
However, like other anticancer agents, flavopiridol (0.5μM) still cytotoxic toward the control group HPASMCs, demonstrating that some therapeutic strategies are needed to reduce the usage of flavopiridol to treat pulmonary hypertension. In the present study, the three assayed CDK9-related negative regulators (LARP7, HEXIM1 and MePCE) were downregulated in isolated PH pulmonary arterial vessels. Furthermore, whether the overexpression of these proteins combined with CDK9 inhibition (flavopiridol) can exert synergistic inhibitory effects toward CDK9-mediated transcriptional elongation to yield a more pronounced suppression of the development of pulmonary hypertension and decrease side effects will be investigated in our further studies.
There were several limitations of the present study. First, due to the technical simplicity and reproducibility advantages, an MCT-induced pulmonary hypertension model was used in our present study, but the differences between MCT models and human pulmonary hypertension should be taken into consideration. MCT not only induces PH but also affects both the right and left ventricles as well as other organs (liver and kidney injuries), which may affect the progression of pulmonary hypertension [38]. In subsequent studies, analyses of pulmonary artery samples from pulmonary hypertension patients may be more convincing to evaluate the associated role of CDK9. Second, flavopiridol is a pan-CDK9 inhibitor and may alter transcription via other mechanisms in addition to CDK9 inhibition or could inhibit other CDKs, although with lower efficacy [35]. Thus,it would be more convincing if a higher specific CDK9 inhibitor (e.g., BAY-1143572) was used in subsequent studies [39]. Third, P-TEFb comprise CDK9 and cycin T (T1, T2a or T2b), and cyclin T1 is the primary CDK9 partner cyclin (approximately 80%) [37]. In addition, cyclin T1 has also been reported to be overexpressed in cancer tissues compared to normal tissues, enhancing transcriptional elongation and promoting tumor malignances [40]. However, whether cyclin T1 is upregulated and synergies with CDK9 upregulation to facilitate the development of pulmonary hypertension requires further investigation.
In summary, CDK9 upregulation and the subsequent enhanced activation of transcriptional elongation, which leads to increased downstream prosurvival and antiapoptotic proteins expression, may be associated with the mechanisms involved in pulmonary artery remodeling and facilitate the progression of pulmonary hypertension. Flavopiridol significantly attenuated the pulmonary artery remodeling, reversed the progression of pulmonary hypertension by inhibiting CDK9-meditated transcription elongation. These findings may provide novel insights in the pathological mechanism of pulmonary hypertension, and CDK9 inhibition, like flavopiridol treatment, should be considered a potential novel therapeutic strategy for pulmonary hypertension.