Introduction
Fibrosis are characterized by excessive proliferation and transformation of fibroblast and extracellular matrix (ECM) deposition. The annual incidence of fibrotic diseases is 4,968 per 100,000 person-years, resulting in a severe burden on patients (Zhao et al., 2020). Fibrotic diseases include multiple organs, such as the liver, kidney, heart, lung, and urinary bladder (Henderson et al., 2020). Moreover, the abnormal activation of myofibroblast triggered by transforming growth factor, platelet-derived growth factor, and fibroblast growth factor is identified as a major alteration in fibrosis (Zhao et al., 2022). Hence, a large number of patients suffer from fibrotic diseases that need more effective therapies indeed.
The fibrotic process is initiated by tissue injury. Moderate injury often leads to a tissue repair process. In contrast, severe or long-term wound-healing strategies can cause fibrotic changes to tissue and organ. The etiologies vary in different situations, including hypertension, myocardial infarction, acute or chronic infection, diabetes, alcohol damage, radiation, and others (Rockey et al., 2015). In the urological system, fibrotic processes are triggered in the kidney, ureter, and urinary bladder. Bladder fibrosis is often caused by partial bladder outlet obstruction (pBOO), cystitis, and radiation (Di et al., 2022). We have investigated that mechanical cues in pBOO, including hydrostatic pressure, fluid shear stress, stretching force, and ECM stiffness, activate the bladder fibrosis process.
Yes-associated protein (YAP) has long been recognized as an intracellular mechanical transducer (Dupont et al., 2011). As an important transcriptional co-activator, YAP is also a crucial downstream effector of the Hippo signaling pathway (Moya and Halder, 2019). YAP can respond to cell geometry, density, and substrate adhesion to promote the progression of fibrotic diseases. YAP has been commonly identified in cell renewal, cell differentiation, epithelial-to-mesenchymal transition, and fibrosis (Tang et al., 2013; Ohgushi et al., 2015; Lin et al., 2018). In addition, YAP has been investigated in many mechanical cues-induced organ remodeling, such as atherosclerosis (Wang et al., 2016), orthodontic tooth movement (Deng et al., 2021), nerve regeneration (Li et al., 2021), and others.
YAP can sense the change of stretch and ECM stiffness in the urinary bladder to promote downstream gene expression. Besides ECM deposition, bladder smooth muscle proliferation is an important pathological change in the compensatory stage of bladder fibrosis (Lai et al., 2019; Chen et al., 2020). Therefore, an RNA transcriptome sequencing (RNA-seq) of YAP-knockdown (YAP-KD) and YAP CUT&Tag of human bladder smooth muscle cell (hBdSMC) were performed to investigate the key genes and regulatory network of smooth muscle proliferation in bladder fibrosis. Chromatin immunoprecipitation sequencing (ChIP-seq) is used to study transcriptional factors and target DNA. The prediction through DNA segments indicates the interaction between transcriptional factors and downstream molecules. The CUT&Tag is an improved ChIP-seq with higher quality and can be conducted with only 105 cells (Kaya-Okur et al., 2019). In fibrosis research, we aim to provide novel insights into YAP-associated smooth muscle proliferation.
Discussion
YAP engages in many biological processes, such as cell proliferation, differentiation, apoptosis, and metabolism. In fibrotic diseases, YAP engages in fibroblast activation (Liu et al., 2015), wound healing process (Dey et al., 2020), and ECM remodeling (Zhang et al., 2022). Importantly, YAP can respond to mechanical cues to promote atherosclerosis, fibrosis, cardiac hypertrophy, muscular dystrophy, and cancer (Panciera et al., 2017). For example, the CRISPR/Cas9-mediated endothelial YAP knockdown attenuated the vascular plaque formation in mice, which indicates the association between YAP and atherosclerosis (Wang et al., 2016). The ECM stiffness triggered TGFβ-YAP signaling to promote kidney fibrosis (Szeto et al., 2016). In addition, ECM remodeling activated cardiac fibroblast and cardiac hypertrophy (Perestrelo et al., 2021). Furthermore, YAP also plays a pivotal role in mechanically sensing the urinary bladder. However, inflammatory responses, epithelial cell responses, smooth muscle cell hypertrophy, and ECM remodeling are all included in bladder fibrosis. Whether YAP functions in these processes are unclarified. Hence, the purpose of our study is to elaborate on the function of YAP of smooth muscle in bladder fibrosis progression.
We first infected the hBdSMCs with AAV in the current study to knock down YAP gene expression. Then RNA-seq was performed to illustrate YAP-regulated gene functions and pathways. The results revealed that YAP might engage in cell cycle regulation. With the highly development of high-throughput sequencing technologies, there comes great attention on multiple omics analyses to clarify human diseases (Yu et al., 2021). Hence, a CUT&Tag sequencing was conducted to identify the genes that directly bind to YAP. In the top 10 predicted genes, Fra-1-regulated transcription was reported to promote the proliferation process of breast cancer (Zhao et al., 2014). Studies demonstrated that the BATF-Jun family interacted with interferon regulatory factor 4 to promote lymphoid development (Li et al., 2012). In the activator protein-1 (AP-1) transcription factor family, JunB functions dually in the cell cycle. JunB was initially recognized as a cell proliferation inhibitor. However, JunB also promotes cell division (Piechaczyk and Farràs, 2008), i,e., JunB is identified as pivotal in angiogenesis (Yoshitomi et al., 2021). Like JunB, c-Jun also belongs to the AP-1 family, which involves many biological processes, such as cell proliferation, survival, apoptosis, and tissue morphogenesis (Meng and Xia, 2011). In kidney fibrosis, G2/M-arrested proximal tubular cells facilitated c-Jun signaling to promote the production of fibrotic cytokines (Yang et al., 2010). YAP often binds to the TEA-binding domain (TEAD) family of transcription enhancers to promote downstream gene expression (Kaan et al., 2017). A recent study demonstrated that TEAD1 promoted vascular smooth muscle cells (VSMCs) through solute carrier family member 5 (SLC1A5), thereby activating mTORC1 signaling to facilitate endothelium formation (Osman et al., 2019).
After interactive matching, 36 DEGs were selected. The sheltered hub genes enriched the cell cycle process, which indicated that YAP promoted hBdSMCs proliferation in response to mechanical stimuli. The PPI network stated the top five hub genes through rankings. Coincidentally, the five hub genes are all down-regulated. Cell division cycle-associated 5 (CDCA5) has been widely studied in human cancer progression. In general, CDCA5 is identified as an oncogene and has a poor prognosis for cancers (Chang et al., 2015). For instance, CDCA5-knockdown inhibited cell proliferation, migration, and clone formation in breast cancer (Hu et al., 2022). The degradation of CDCA5 also inhibits prostate cancer progression (Luo et al., 2021). CDCA5 regulates cell proliferation through various signaling. Recent research illustrated that CDCA5 activated prostate cancer and colorectal cancer cell proliferation via ERK signaling pathway (Shen et al., 2019; Ji et al., 2021). In addition, the ablation of CDCA5 inhibited gastric cancer cell proliferation via downregulating Cyclin E1 expression (Zhang et al., 2018).
Centromere Protein A (CENPA) is highly correlated with cell proliferation. The centromere is a chromatin structure that provides an assembly site for cell machinery, which is essential in cell proliferation and survival (Fukagawa and Earnshaw, 2014). Mechanistically, CENPA is assembled into the centromeric chromatin in the cell cycle to the following cell cycle and generation (Wong et al., 2020). Studies demonstrated that the expression of CENPA in cardiac progenitor cells (CPCs) decreased along with aging. The expression level of CENPA is relatively higher in the early stage of life, thereby sustaining cell proliferation, inhibiting senescence, and triggering CPCs differentiation (McGregor et al., 2014). The ablation of CENPA inhibited cell proliferation in ovarian cancer (Han et al., 2021). Interestingly, circular RNAs (circRNAs), defined as crucial cancer regulators, decreased the expression of FOXM1 and promoted the expression of CENPA and CENPB to facilitate cell cycle progression (Cheng et al., 2019).
DTL is identified as CUL4-DDB1 associated factors (DCAFs), engaged in many tumorigenesis processes. Studies revealed that DTL enhanced the proliferation and migration of cancer cells in nude mice (Cui et al., 2019). In addition, the increase of DTL indicated a poor prognosis in malignant manners of bladder cancer through the mTOR/Akt signaling cascades (Luo et al., 2022). Similarly, the non-structural maintenance of chromosomes condensing I complex subunit H (NCAPH) also facilitated cell proliferation, migration, invasion, and epithelial-to-mesenchymal transition (EMT) of cancer (Kim et al., 2020; Wang et al., 2020). And some microRNAs also target NCAPH to promote the degradation of β-catenin to reduce cancer stem cell maintenance (Wang et al., 2020). The fifth hub gene, the base excision repair enzyme NEIL3, plays a vital role in many biological processes, including fibrosis, lipid metabolism, tumorigenesis, and neurogenesis. For instance, in NEIL3−/− heart ruptured mice, the fibroblasts and myofibroblasts increased significantly, indicating that NEIL3-dependent regulation of DNA methylation affected the fibroblast proliferation and the ECM modulation (Olsen et al., 2017). Interestingly, we have found that epigenetics alterations in cell-free DNA genome were widely distributed in multiple diseases, which might be critical in early diagnosis of fibrosis and cancer disease (Yu et al., 2020). In the smooth muscle remodeling process, the depletion of DNA glycosylase NEIL3 promoted differentiation of aortic VSMCs through the Akt signaling pathway (Quiles-Jiménez et al., 2021). NEIL3 has also been reported to mediate the lipid metabolism and macrophage function in myocardial infarction (Skarpengland et al., 2016).
Collectively, the hub genes are enriched in the processes of cell proliferation and cell survival. The question is how smooth muscle proliferation behaves in bladder fibrosis. pBOO was caused by bladder wall inflammation, hypertrophy, and fibrosis (Siregar et al., 2022). Studies demonstrated that pBOO-induced bladder fibrosis was attributed to fibrosis, smooth muscle cell proliferation and hypertrophy, and urothelium proliferation (Qiao et al., 2018). In female pBOO rats, the bladder smooth muscle progresses in hematoxylin and eosin staining and is confirmed by increased bladder mass and thickness increase (Metcalfe et al., 2010). The bladder wall thickening functions in compensatory mechanisms against pathological mechanical forces in the urinary bladder, which ultimately results in a fibrotic bladder with low capacity and high pressure. Therefore, early intervention of pBOO is necessary, and knowing how to regulate these hub genes’ functions might provide novel insights into preventing pBOO-induced fibrosis. In addition, a recent study demonstrated that computational framework for analyzing multi-omics profiles provided a novel direction for clinical diagnosis of many diseases (Yu et al., 2019). Combined analyses based on well-established tools may validate the results further.
Our study inevitably has some limitations. Although the CUT&Tag sequencing is more reliable than the common ChIP-seq, the low repeatability is still a common claw that cannot be solved to date. Hence, only two samples (Experiment and IgG) were applied for CUT&Tag sequencing. Furthermore, since bladder fibrosis is associated with several cell types, including urothelial cells, smooth muscle cells, and fibroblasts, the results from bladder smooth muscle cells can partly reflect the possible therapeutic targets. Further research on other cell types is needed.