Both protein kinase C (PKC) and reactive oxygen species (ROS) are well-known signaling messengers cross-talking with each other to activate mitogen-activated protein kinases (MAPKs) for progression of hepatocellular carcinoma (HCC). However, the underlying mechanisms are not well elucidated. Especially, whether mitochondrial ROS (mtROS) is involved and how it triggers MAPK signaling are intriguing. In this study, we found mtROS generation and phosphorylation of MAPKs were mediated by PKCδ in HCCs treated with the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Heat shock protein 60 (HSP60), one of the chaperones in mitochondria was the major protein oxidized in TPA-treated HCCs. Moreover, depletion of HSP60 or expression of HSP60 cysteine mutant prevented TPA-induced phosphorylation of MAPKs. To delineate how HSP60 mediated MAPK activation, the role of Raf kinase inhibitor protein (RKIP), a negative regulator of MAPK, was investigated. TPA dissociated RKIP from HSP60 in both mitochondria and cytosol, concurrently with translocation of HSP60 and MAPK from mitochondria to cytosol, which was associated with robust phosphorylation of MAPKs in the cytosol. Moreover, TPA induced opposite phenotypical changes of HCCs, G1 cell cycle arrest, and cell migration, which were prevented by mtROS scavengers and depletion of PKCδ and HSP60. Consistently, TPA increased the migration-related genes, hydrogen peroxide inducible clone5, matrix metalloproteinase-1/3, lamininγ2, and suppressed the cell cycle regulator cyclin E1 (CCNE1) via PKCδ/mtROS/HSP60/MAPK-axis. Finally, c-jun and c-fos were required for TPA-induced expression of the migration-related genes and a novel microRNA, miR-6134, was responsible for TPA-induced suppression of CCNE1. In conclusion, PKCδ cross-talked with mtROS to trigger HSP60 oxidation for release of RKIP to activate MAPK, regulating gene expression for migration, and G1 cell cycle arrest in HCC. Targeted therapy aiming at key players like PKCδ, RKIP, and HSP60 is promising for preventing HCC progression.

SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established.

Phenylacetylglutamine (PAG), a gut microbiota metabolite, is associated with cardiovascular diseases. Arterial stiffness (AS), which is a marker of aging-associated vascular diseases, is an independent risk factor for cardiovascular morbidity and mortality. This study aimed to assess the correlation between serum PAG levels and AS in kidney transplantation (KT) patients, potentially uncovering new insights into the cardiovascular risks in this population. In this study, 100 KT patients were included. Carotid–femoral pulse wave velocity (cfPWV) was measured, and patients with cfPWV > 10 m/s were categorized as the AS group. Serum PAG levels were assessed using liquid chromatography–tandem mass spectrometry. Thirty KT patients (30.0%) exhibited AS, with higher percentages of diabetes mellitus, older age, and elevated levels of systolic blood pressure, serum fasting glucose, and PAG than the control group. After adjusting for factors significantly associated with AS by multivariate logistic regression analysis, serum PAG, age, fasting glucose levels, and systolic blood pressure were independent factors associated with AS. Furthermore, PAG levels had a negative correlation with the estimated glomerular filtration rate and a positive correlation with cfPWV values. Serum PAG levels are positively associated with cfPWV values and are a biomarker of AS in KT patients.

Objectives: Osteocalcin, a protein from osteoblasts, affects bone mineralization and turnover. This study evaluates the association between fasting serum osteocalcin and bone mineral density (BMD) in renal transplant recipients. Materials and Methods: This study recruited 66 renal transplant recipients. We analyzed blood biochemistry studies from fasting blood samples. The serum osteocalcin levels were measured using a commercial enzyme immunoassay kit. We measure BMD by dual-energy X-ray absorptiometry in lumbar vertebrae (L2–L4). By the World Health Organization classification, we group recipients into three groups: normal, osteopenia, and osteoporosis. Results: Of the renal transplant recipients, 8 patients (12.1%) were osteoporosis, and 28 patients (42.4%) were osteopenia. From normal to osteoporosis groups, the osteoporosis group has highest serum osteocalcin (P < 0.001), alkaline phosphatase (P = 0.005), lowest body mass index (P = 0.015), and body weight (P = 0.008). Females had lower lumbar BMD than males among recruited renal transplant recipients (P = 0.023). In the multivariate forward stepwise linear regression analysis, body weight (adjusted R ² change = 0.138; P = 0.010), and logarithmically transformed osteocalcin (log-osteocalcin; adjusted R² change = 0.131; P = 0.012) can predict lumbar BMD in the renal transplant recipients. Conclusion: Our study showed that fasting serum osteocalcin concentration was negatively correlated with the lumbar BMD in renal transplant recipients.

The primary role of myostatin is to negatively regulate skeletal muscle growth. The gait speed is a noninvasive, reliable parameter that predicts cardiovascular risk and mortality. This study evaluated the relationship between serum myostatin concentrations and gait speeds in patients who had undergone kidney transplantation (KT). A total of 84 KT recipients were evaluated. A speed of less than 1.0 m/s was categorized into the low gait speed group. We measured serum myostatin concentrations with a commercial enzyme-linked immunosorbent assay. KT recipients in the low gait speed group had significantly older age, as well as higher body weight, body mass index (BMI), skeletal muscle index, serum triglyceride levels, glucose levels, and blood urea nitrogen levels, lower estimated glomerular filtration rates and serum myostatin levels, a higher percentage of steroid use, and a lower proportion of mycophenolate mofetil use. Multivariable logistic regression analysis revealed that lower myostatin levels and lower frequency of mycophenolate mofetil use were independently associated with low gait speed. In multivariable stepwise linear regression analysis, myostatin levels were positively correlated with gait speeds, and age and BMI were negatively correlated with gait speeds. In the study, serum myostatin levels were significantly lower in the low gait speed group. Subjects in the low gait speed group also had greater BMI and older age.