Protein phosphatase 5 (PP5), a serine/threonine phosphatase, has a wide range of biological functions and exhibits elevated expression in tumor cells. We previously reported that pp5-deficient mice have altered ataxia-telangiectasia mutated (ATM)-mediated signaling and function. However, this regulation was likely indirect, as ATM is not a known PP5 substrate. In the current study, we found that pp5-deficient mice are hypersensitive to genotoxic stress. This hypersensitivity was associated with the marked up-regulation of the tumor suppressor tumor protein p53 and its downstream targets cyclin-dependent kinase inhibitor 1A (p21), MDM2 proto-oncogene (MDM2), and phosphatase and tensin homolog (PTEN) in pp5-deficient tissues and cells. These observations suggested that PP5 plays a role in regulating p53 stability and function. Experiments conducted with p53^+/−pp5^+/− or p53^+/−pp5^−/− mice revealed that complete loss of PP5 reduces tumorigenesis in the p53^+/− mice. Biochemical analyses further revealed that PP5 directly interacts with and dephosphorylates p53 at multiple serine/threonine residues, resulting in inhibition of p53-mediated transcriptional activity. Interestingly, PP5 expression was significantly up-regulated in p53-deficient cells, and further analysis of pp5 promoter activity revealed that p53 strongly represses PP5 transcription. Our results suggest a reciprocal regulatory interplay between PP5 and p53, providing an important feedback mechanism for the cellular response to genotoxic stress.

High levels of alcohol intake alter brain gene expression and can produce long-lasting effects. FK506-binding protein 51 (FKBP51) encoded by Fkbp5 is a physical and cellular stress response gene and has been associated with alcohol consumption and withdrawal severity. Fkbp5 has been previously linked to neurite outgrowth and hippocampal morphology, sex differences in stress response, and epigenetic modification. Presently, primary cultured Fkbp5 KO and WT mouse neurons were examined for neurite outgrowth and mitochondrial signal with and without alcohol. We found neurite specification differences between KO and WT; particularly, mesh-like morphology was observed after alcohol treatment and confirmed higher MitoTracker signal in cultured neurons of Fkbp5 KO compared to WT at both naive and alcohol-treated conditions. Brain regions that express FKBP51 protein were identified, and hippocampus was confirmed to possess a high level of expression. RNA-seq profiling was performed using the hippocampus of naïve or alcohol-injected (2 mg EtOH/Kg) male and female Fkbp5 KO and WT mice. Differentially expressed genes (DEGs) were identified between Fkbp5 KO and WT at baseline and following alcohol treatment, with female comparisons possessing a higher number of DEGs than male comparisons. Pathway analysis suggested that genes affecting calcium signaling, lipid metabolism, and axon guidance were differentially expressed at naïve condition between KO and WT. Alcohol treatment significantly affected pathways and enzymes involved in biosynthesis (Keto, serine, and glycine) and signaling (dopamine and insulin receptor), and neuroprotective role. Functions related to cell morphology, cell-to-cell signaling, lipid metabolism, injury response, and post-translational modification were significantly altered due to alcohol. In summary, Fkbp5 plays a critical role in the response to acute alcohol treatment by altering metabolism and signaling-related genes.

Background and aims: Previously, we found that FK506 binding protein 51 (Fkbp51) knockout (KO) mice resist high fat diet-induced fatty liver and alcohol-induced liver injury. The aim of this research is to identify the mechanism ofFkbp51in liver injury.Methods: Carbon tetrachloride (CCl₄)-induced liver injury was compared betweenFkbp51KO and wild type (WT) mice. Step-wise and in-depth analyses were applied, including liver histology, biochemistry, RNA-Seq, mitochondrial respiration, electron microscopy, and molecular assessments. The selective FKBP51 inhibitor (SAFit2) was tested as a potential treatment to ameliorate liver injury.Results: Fkbp51knockout mice exhibited protection against liver injury, as evidenced by liver histology, reduced fibrosis-associated markers and lower serum liver enzyme levels. RNA-seq identified differentially expressed genes and involved pathways, such as fibrogenesis, inflammation, mitochondria, and oxidative metabolism pathways and predicted the interaction of FKBP51, Parkin, and HSP90. Cellular studies supported co-localization of Parkin and FKBP51 in the mitochondrial network, and Parkin was shown to be expressed higher in the liver of KO mice at baseline and after liver injury relative to WT. Further functional analysis identified that KO mice exhibited increased ATP production and enhanced mitochondrial respiration. KO mice have increased mitochondrial size, increased autophagy/mitophagy and mitochondrial-derived vesicles (MDV), and reduced reactive oxygen species (ROS) production, which supports enhancement of mitochondrial quality control (MQC). Application of SAFit2, an FKBP51 inhibitor, reduced the effects of CCl₄-induced liver injury and was associated with increased Parkin, pAKT, and ATP production.Conclusions: Downregulation of FKBP51 represents a promising therapeutic target for liver disease treatment.Graphical

This study aimed to investigate the role of protein phosphatase 5 (PP5) on bone and cartilage development using both in vivo and in vitro approaches. Six- to 8-week- old male PP5 knockout mice (KO) and their wild-type (WT) littermate controls were randomly selected for this study, and their body weights and bone (femur) lengths were measured. Micro-computed tomography scanning (Micro-CT) was performed to determine femoral bone density and micro-architecture. Mesenchymal stem cells (MSCs) isolated from bone marrow were used to examine the effects of PP5 on osteogenesis in vitro. Whole-mount Alcian blue and Alizarin red staining were used to detect cartilage formation in newborn vertebrae, limbs, and feet. Hematoxylin and eosin (H&E) staining was performed to determine growth plate thickness. Real-time PCR analysis, western blotting, and immunohistochemistry were used to detect the expression of genes and proteins in bone marrow-derived MSCs as well as in bone and cartilage tissues. The results showed PP5 KO mice exhibited significantly reduced body weight and shorter femur length compared to WT controls. The KO mice also had significantly higher volumetric bone mineral density (BMD), trabecular bone volume, and cortical thickness in the femur. The deficiency of PP5 significantly enhanced the formation of cartilage in vertebrae, limbs, and feet. In addition, KO mice possessed a wider distal femur growth plates containing significantly more chondrocytes than WT mice. Furthermore, higher expressions of several cartilage-specific genes were observed in the articular cartilage of PP5 KO mice. Immunohistochemical labeling of growth plates demonstrated that phospho-PPARγ, Runx1, and Runx2 levels were considerably higher in the KO mice. In conclusion, PP5 is a significant negative regulator on the regulation of bone and cartilage development.