The bone remodelling relies on a dynamic balance between bone formation and resorption, mediated by osteoblasts and osteoclasts, respectively. Under certain stimuli, osteoprogenitor cells may differentiate into premature osteoblasts and further into mature osteoblasts. This process is marked by increased alkaline phosphatase (ALP) activity and mineralized nodule formation. In this study, we induced osteoblast differentiation in mouse osteoprogenitor MC3T3-E1 cells and divided the process into three stages. In the first stage (day 3), the MC3T3-E1 cell under osteoblast differentiation did not express ALP or deposit mineralized nodule. In the second stage, the MC3T3-E1 cell expressed ALP but did not form mineralized nodule. In the third stage, the MC3T3-E1 cell had ALP activity and formed mineralized nodule. In present study, we focused on morphological and proteomic changes of MC3T3-E1 cells in the early stage of osteoblast differentiation - a period when premature osteoblasts transform into mature osteoblasts. We found that mean cell area and mean stress fiber density were increased in this stage due to enhanced cell spreading and decreased cell proliferation. We further analyzed the proteins in the signaling pathway of regulation of cytoskeleton using proteomic approach and found upregulation of IQGAP1, gelsolin, moesin, radixin, and Cfl1. After analyzing the focal adhesion signaling pathway, we found the upregulation of FLNA, LAMA1, LAMA5, COL1A1, COL3A1, COL4A6, and COL5A2 as well as the downregulation of COL4A1, COL4A2, and COL4A4. In conclusion, the signaling pathway of regulation of cytoskeleton and focal adhesion play critical roles in regulating cell spreading and actin skeleton formation in the early stage of osteoblast differentiation.

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PML protein plays important roles in regulating cellular homeostasis. It forms PML nuclear bodies (PML-NBs) that act like nuclear relay stations and participate in many cellular functions. In this study, we have examined the proteome of mouse embryonic fibroblasts (MEFs) derived from normal (PML+/+) and PML knockout (PML−/−) mice. The aim was to identify proteins that were differentially expressed when MEFs were incapable of producing PML. Using comparative proteomics, total protein were extracted from PML−/− and PML+/+ MEFs, resolved by two dimensional electrophoresis (2-DE) gels and the differentially expressed proteins identified by LC-ESI-MS/MS. Nine proteins (PML, NDRG1, CACYBP, CFL1, RSU1, TRIO, CTRO, ANXA4 and UBE2M) were determined to be down-regulated in PML−/− MEFs. In contrast, ten proteins (CIAPIN1, FAM50A, SUMO2 HSPB1 NSFL1C, PCBP2, YWHAG, STMN1, TPD52L2 and PDAP1) were found up-regulated. Many of these differentially expressed proteins play crucial roles in cell adhesion, migration, morphology and cytokinesis. The protein profiles explain why PML−/− and PML+/+ MEFs were morphologically different. In addition, we demonstrated PML−/− MEFs were less adhesive, proliferated more extensively and migrated significantly slower than PML+/+ MEFs. NDRG1, a protein that was down-regulated in PML−/− MEFs, was selected for further investigation. We determined that silencing NDRG1expression in PML+/+ MEFs increased cell proliferation and inhibited PML expression. Since NDRG expression was suppressed in PML−/− MEFs, this may explain why these cells proliferate more extensively than PML+/+ MEFs. Furthermore, silencing NDRG1expression also impaired TGF-β1 signaling by inhibiting SMAD3 phosphorylation.

Adipose tissues are essential for actively regulating systemic energy balance, glucose homeostasis, immune responses, reproduction, and longevity. Adipocytes maintain dynamic metabolic needs and possess heterogeneity in energy storage and supply. Overexpansion of adipose tissue, especially the visceral type, is a high risk for diabetes and other metabolic diseases. Changes in adipocytes, hypertrophy or hyperplasia, contribute to the remodeling of obese adipose tissues, accompanied by abundant immune cell accumulation, decreased angiogenesis, and aberrant extracellular matrix deposition. The process and mechanism of adipogenesis are well known, however, adipose precursors and their fate decision are only being defined with recent information available to decipher how adipose tissues generate, maintain, and remodel. Here, we discuss the key findings that identify adipose precursors phenotypically, with special emphasis on the intrinsic and extrinsic signals in instructing and regulating the fate of adipose precursors under pathophysiological conditions. We hope that the information in this review lead to novel therapeutic strategies to combat obesity and related metabolic diseases.