human coronary artery smcs (hcasmcs)  (Lonza)

Journal: The Journal of Biological Chemistry

Article Title: Declining activity of serum response factor in aging aorta in relation to aneurysm progression

doi: 10.1016/j.jbc.2025.108400

Figure Lengend Snippet: MYOCD/SRF remodels Hippo. To understand YAP/TAZ activation in i8-SRF-KO aorta, we overlapped YAP/TAZ regulators (171 proteins) from a recent review with the differentially expressed proteins in i8-SRF-KO versus control aorta (729 proteins) to identify an overlap of 27 proteins ( A ). YAP/TAZ activators that were increased and inhibitors that were reduced (i8-SRF-KO vs. Ctrl) were highlighted with their respective protein names in black. Subsequently, 11 out of 27 changes were expected to activate YAP/TAZ whereas the remainder were expected to inhibit YAP/TAZ. Because core Hippo kinases were not recovered in our mass spectroscopy experiment, we next overlapped core Hippo constituents with a previous RNA-seq experiment (n = 4 experiments were cell were treated with either Ad-CMV-null or Ad-CMV-MYOCD) where MYOCD was overexpressed, uncovering significant increases of MOB1A , LATS1 , and LATS2 ( B ). Moreover, both SRF and MYOCD correlated with LATS2 at the mRNA level across human arteries ( C ). We therefore hypothesized that MYOCD/SRF may control the LATS2 protein level. Western blotting using lysates from control and i8-SRF-KO aortae revealed reduction of a major Lats2 band migrating at 240 kDa ( D , summarized data in E , n = 6 mice in each group) following SRF depletion. Lats1 ( D ), and Slmap (major isoform at 40 kDa, D ), included to allow for comparison with the mass spectroscopy in A , were not significantly reduced ( D and F ). HSP90, H3, and proteins remaining on the gel after transfer were included as loading controls. A Lats2 band migrating at 240 kDa, essentially eliminated by a LATS2-targeted siRNA (siLATS2c), was also detected in cultured smooth muscle cells from the human coronary artery following MYOCD transduction ( G , n = 3 culture wells). In a larger experiment, overexpression of MYOCD in human SMCs increased LATS2 >50-fold ( H , I , n = 6 culture wells in two separate experiments). This was associated with an increased phospho-MOB1 level ( H and J ) and an increased P-YAP over total YAP ratio ( H and K ). Taken together, this suggests that MYOCD/SRF promotes the activity of Hippo via phospho-MOB1 and LATS2 to suppress YAP/TAZ activity; consequently, deletion of SRF in the aorta is expected to release YAP/TAZ from a critical inhibitory influence from Hippo. As prior work demonstrated that YAP/TAZ are important upstream regulators of MYOCD, we propose that the effect of MYOCD/SRF on Hippo represents an inhibitory and homeostatic feedback loop as depicted schematically in L. Lats2, large tumor suppressor kinase 2; MYOCD, myocardin; SMC, smooth muscle cell; SRF, serum response factor; TAZ, WW domain containing transcription regulator 1; YAP, Yes associated transcriptional regulator.

Article Snippet: Human coronary artery SMCs were obtained from Thermo Fisher Scientific (C-017-5C, Gibco) and cultured in Human Vascular Smooth Muscle Cell Basal Medium (M231500, Thermo Fisher Scientific) supplemented with 5% smooth muscle growth supplement (S00725) and PEST (50 U/ml penicillin and 50 μg/ml streptomycin, Biochrom, A 2212).

Techniques: Activation Assay, Control, Mass Spectrometry, RNA Sequencing, Western Blot, Comparison, Cell Culture, Transduction, Over Expression, Activity Assay