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Cetrullo S.,University of Bologna | D'Adamo S.,University of Bologna | Tantini B.,University of Bologna | Borzi R.M.,Laboratorio Of Immunoreumatologia E Rigenerazione Tessutale | And 2 more authors.
Critical Reviews in Eukaryotic Gene Expression | Year: 2015

Cells adapt their metabolism and activities in response to signals from their surroundings, and this ability is essential for their survival in the face of environmental changes. In mammalian tissues a deficit of these mechanisms is commonly associated with cellular aging and degenerative diseases related to aging, such as cardiovascular disease, cancer, immune system decline, and neurological pathologies. Several proteins have been identified as able to respond directly to energy, nutrient, and growth factor levels and stress stimuli in order to mediate adaptations in the cell. Many of these proteins are enzymes that positively or negatively modulate the autophagic process. This review focuses on biochemical mechanisms involving enzymes—specifically, mTOR, AMPK, and Sirt1—that are currently considered important for these adaptive responses, providing an overview of the interactions of the main players in this process. © 2015 Begell House, Inc.

Guidotti S.,Laboratorio Of Immunoreumatologia E Rigenerazione Tessutale | Guidotti S.,University of Bologna | Minguzzi M.,Laboratorio Of Immunoreumatologia E Rigenerazione Tessutale | Minguzzi M.,University of Bologna | And 10 more authors.
PLoS ONE | Year: 2015

Introduction Recent evidence suggests that GSK3 activity is chondroprotective in osteoarthritis (OA), but at the same time, its inactivation has been proposed as an anti-inflammatory therapeutic option. Here we evaluated the extent of GSK3β inactivation in vivo in OA knee cartilage and the molecular events downstream GSK3β inactivation in vitro to assess their contribution to cell senescence and hypertrophy. Methods In vivo level of phosphorylated GSK3β was analyzed in cartilage and oxidative damage was assessed by 8-oxo-deoxyguanosine staining. The in vitro effects of GSK3β inactivation (using either LiCl or SB216763) were evaluated on proliferating primary human chondrocytes by combined confocal microscopy analysis of Mitotracker staining and reactive oxygen species (ROS) production (2',7'-dichlorofluorescin diacetate staining). Downstream effects on DNA damage and senescence were investigated by western blot (ãH2AX, GADD45β and p21), flow cytometric analysis of cell cycle and light scattering properties, quantitative assessment of senescence associated β galactosidase activity, and PAS staining. Results In vivo chondrocytes from obese OA patients showed higher levels of phosphorylated GSK3β, oxidative damage and expression of GADD45β and p21, in comparison with chondrocytes of nonobese OA patients. LiCl mediated GSK3β inactivation in vitro resulted in increased mitochondrial ROS production, responsible for reduced cell proliferation, S phase transient arrest, and increase in cell senescence, size and granularity. Collectively, western blot data supported the occurrence of a DNA damage response leading to cellular senescence with increase in ãH2AX, GADD45β and p21. Moreover, LiCl boosted 8-oxo-dG staining, expression of IKKα and MMP-10. Conclusions In articular chondrocytes, GSK3β activity is required for the maintenance of proliferative potential and phenotype. Conversely, GSK3β inactivation, although preserving chondrocyte survival, results in functional impairment via induction of hypertrophy and senescence. Indeed, GSK3β inactivation is responsible for ROS production, triggering oxidative stress and DNA damage response. © 2015 Guidotti et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Guidotti S.,Laboratorio Of Immunoreumatologia E Rigenerazione Tessutale | Guidotti S.,University of Bologna | Facchini A.,University of Bologna | Platano D.,Laboratorio Of Immunoreumatologia E Rigenerazione Tessutale | And 15 more authors.
Stem Cells and Development | Year: 2013

The molecular mechanisms underlying spermine osteo-inductive activity on human adipose-derived stem cells (ASCs) grown in 3-dimensional (3D) cultures were investigated. Spermine belongs to the polyamine family, naturally occurring, positively charged polycations that are able to control several cellular processes. Spermine was used at a concentration close to that found in platelet-rich plasma (PRP), an autologous blood product containing growth and differentiation factors, which has recently become popular in in vitro and in vivo bone healing and engineering. Adipose tissue was surgically obtained from the hypodermis of patients undergoing hip arthroplasty. Patient age negatively affected both ASC yield and ASC ability to form 3D constructs. ASC 3D cultures were seeded in either non differentiating or chondrogenic conditions, with or without the addition of 5 μM spermine to evaluate its osteogenic potential across 1, 2, and 3 weeks of maturation. Osteogenic medium was used as a reference. The effects of the addition of spermine on molecular markers of osteogenesis, at both gene and protein level, and mineralization were evaluated. The effects of spermine were temporally defined and responsible for the progression from the early to the mature osteoblast differentiation phases. Spermine initially promoted gene and protein expression of Runx-2, an early marker of the osteoblast lineage; then, it increased β-catenin expression and activation, which led to the induction of Osterix gene expression, the mature osteoblast commitment factor. The finding that spermine induces ASC to differentiate toward mature osteoblasts supports the use of these easily accessible mesenchymal stem cells coupled with PRP for orthopedic applications. © Copyright 2013, Mary Ann Liebert, Inc. 2013.

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