Senescent cells secrete senescence-associated secretory phenotype (SASP) proteins to carry out several functions, such as sensitizing surrounding cells to senesce; immunomodulation; impairing or fostering cancer growth; and promoting tissue development. Identifying secreted factors that achieve such tasks is a challenging issue since the profile of secreted proteins depends on genotoxic stress and cell type. Currently, researchers are trying to identify common markers for SASP. The present investigation compared the secretome composition of five different senescent phenotypes in two different cell types: bone marrow and adipose mesenchymal stromal cells (MSC). We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation, and replicative exhaustion. We took advantage of LC-MS/MS proteome identification and subsequent gene ontology (GO) evaluation to perform an unbiased analysis (hypothesis free manner) of senescent secretomes. GO analysis allowed us to distribute SASP components into four classes: extracellular matrix/cytoskeleton/cell junctions; metabolic processes; ox-redox factors; and regulators of gene expression.We used Ingenuity Pathway Analysis (IPA) to determine common pathways among the different senescent phenotypes. This investigation, along with identification of eleven proteins that were exclusively expressed in all the analyzed senescent phenotypes, permitted the identification of three key signaling paths: MMP2 - TIMP2; IGFBP3 - PAI-1; and Peroxiredoxin 6 - ERP46 - PARK7 - Cathepsin D - Major vault protein. We suggest that these paths could be involved in the paracrine circuit that induces senescence in neighboring cells and may confer apoptosis resistance to senescent cells.

Cellular senescence is the permanent arrest of cell cycle, physiologically related to aging and aging-associated diseases. Senescence is also recognized as a mechanism for limiting the regenerative potential of stem cells and to protect cells from cancer development. The senescence program is realized through autocrine/paracrine pathways based on the activation of a peculiar senescence-associated secretory phenotype (SASP). We show here that conditioned media (CM) of senescent mesenchymal stem cells (MSCs) contain a set of secreted factors that are able to induce a full senescence response in young cells. To delineate a hallmark of stem cells SASP, we have characterized the factors secreted by senescent MSC identifying insulin-like growth factor binding proteins 4 and 7 (IGFBP4 and IGFBP7) as key components needed for triggering senescence in young MSC. The pro-senescent effects of IGFBP4 and IGFBP7 are reversed by single or simultaneous immunodepletion of either proteins from senescent-CM. The blocking of IGFBP4/7 also reduces apoptosis and promotes cell growth, suggesting that they may have a pleiotropic effect on MSC biology. Furthermore, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in young MSC. Collectively, these results suggest the occurrence of novel-secreted factors regulating MSC cellular senescence of potential importance for regenerative medicine and cancer therapy.

A sharp definition of what a senescent cell is still lacking since we do not have in depth understanding of mechanisms that induce cellular senescence. In addition, senescent cells are heterogeneous, in that not all of them express the same genes and present the same phenotype. To further clarify the classification of senescent cells, hints may be derived by the study of cellular metabolism, autophagy and proteasome activity. In this scenario, we decided to study these biological features in senescence of Mesenchymal Stromal Cells (MSC). These cells contain a subpopulation of stem cells that are able to differentiate in mesodermal derivatives (adipocytes, chondrocytes, osteocytes). In addition, they can also contribute to the homeostatic maintenance of many organs, hence, their senescence could be very deleterious for human body functions.We induced MSC senescence by oxidative stress, doxorubicin treatment, X-ray irradiation and replicative exhaustion. The first three are considered inducers of acute senescence while extensive proliferation triggers replicative senescence also named as chronic senescence. In all conditions, but replicative and high IR dose senescence, we detected a reduction of the autophagic flux, while proteasome activity was impaired in peroxide-treated and irradiated cells. Differences were observed also in metabolic status. In general, all senescent cells evidenced metabolic inflexibility and prefer to use glucose as energy fuel. Irradiated cells with low dose of X-ray and replicative senescent cells show a residual capacity to use fatty acids and glutamine as alternative fuels, respectively. Our study may be useful to discriminate among different senescent phenotypes.