The CDK9–cyclin T1 complex mediates saturated fatty acid–induced vascular calcification by inducing expression of the transcription factor CHOP

Yuji Shiozaki,Kayo Okamura,Shohei Kohno,Audrey L Keenan,Kristina Williams,Xiaoyun Zhao,Wallace S Chick,Shinobu Miyazaki-Anzai,Makoto Miyazaki

doi:10.1074/jbc.ra118.004706

Abstract

Vascular calcification (or mineralization) is a common complication of chronic kidney disease (CKD) and is closely associated with increased mortality and morbidity rates. We recently reported that activation of the activating transcription factor 4 (ATF4) pathway through the saturated fatty acid (SFA)-induced endoplasmic reticulum (ER) stress response plays a causative role in CKD-associated vascular calcification. Here, using mouse models of CKD, we 1) studied the contribution of the proapoptotic transcription factor CCAAT enhancer–binding protein homologous protein (CHOP) to CKD-dependent medial calcification, and 2) we identified an additional regulator of ER stress–mediated CHOP expression. Transgenic mice having smooth muscle cell (SMC)–specific CHOP expression developed severe vascular apoptosis and medial calcification under CKD. Screening of a protein kinase inhibitor library identified 16 compounds, including seven cyclin-dependent kinase (CDK) inhibitors, that significantly suppressed CHOP induction during ER stress. Moreover, selective CDK9 inhibitors and CRISPR/Cas9-mediated CDK9 reduction blocked SFA-mediated induction of CHOP expression, whereas inhibitors of other CDK isoforms did not. Cyclin T1 knockout inhibited SFA-mediated induction of CHOP and mineralization, whereas deletion of cyclin T2 and cyclin K promoted CHOP expression levels and mineralization. Of note, the CDK9–cyclin T1 complex directly phosphorylated and activated ATF4. These results demonstrate that the CDK9–cyclin T1 and CDK9–cyclin T2/K complexes have opposing roles in CHOP expression and CKD-induced vascular calcification. They further reveal that the CDK9–cyclin T1 complex mediates vascular calcification through CHOP induction and phosphorylation-mediated ATF4 activation.

Highlights

Vascular calcification is a common complication of chronic kidney disease (CKD) and is closely associated with increased mortality and morbidity rates
We recently reported that activation of the activating transcription factor 4 (ATF4) pathway through the saturated fatty acid (SFA)-induced endoplasmic reticulum (ER) stress response plays a causative role in CKD-associated vascular calcification
We examined whether CHOP induction solely induces medial calcification in CKD, and we identified a novel pathway that contributes to ER stress-induced CHOP expression and vascular calcification

Summary

Results

To study the SMC-specific role of CHOP in regulating medial calcification, we generated CHOP conditional transgenic (loxTG) mice similar to the ATF4 loxTG mice that we previously reported [11]. Only compounds inhibiting CDK9, including AT7519 (lane 3), flavopiridol (lane 4), dinaciclib (lane 5), AZD5438 (lane 7), CAY10574 (lane 10), and LDC000067 (lane 11) reduced levels of CHOP induced by C18:0 (Fig. 2C), suggesting that CDK9 regulates CHOP expression. CDK9-specific inhibitor treatment reduced levels of CHOP protein and mRNA (Fig. 2, D and E), whereas other ER stress markers such as ATF4 (Fig. 2D), sXBP-1 (Fig. 2F), BiP (Fig. 2G), and ATF3 (data not shown) were not affected. CDK9 haplo-insufficiency blocked C18:0-mediated CHOP but not other ER stress effectors such as BiP and sXBP1, consistent with treatment with CDK9-specific inhibitors (Fig. 3, C–F). These data suggest that CDK9 selectively regulates CHOP induction under ER stress.

A Rosa26 Locus

Discussion

Experimental procedures