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Citation: Cell Death and Disease (2013) 4, e911; doi:10.1038/cddis.2013.445

Web End =www.nature.com/cddis

V Severino1,2,3,10, N Alessio4,10, A Farina5, A Sandomenico2,3, M Cipollaro4, G Peluso6,7, U Galderisi*,4,6,8,9 and A Chambery*,1,3

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.

Cell Death and Disease (2013) 4, e911; doi:http://dx.doi.org/10.1038/cddis.2013.445

Web End =10.1038/cddis.2013.445 ; published online 7 November 2013

Subject Category: Experimental Medicine

Senescence is the permanent cell cycle arrest that leads to the loss of cellular functions over time. Several lines of evidence correlate cellular senescence to aging, as it largely contributes to reduce tissue functions and renewal. On the other side, it is also believed to promote protective anticancer mechanisms leading to tumor growth arrest.1,2

Senescence of stem cells, such as mesenchymal stem cells (MSCs), could be very deleterious as it can greatly impair tissue homeostasis and repair. MSC are pluripotent cells that reside in postnatal organs and tissues, and can differentiate into bone, cartilage and fat. Owing to their supporting role in hematopoiesis and in the homeostatic maintenance of many organs and tissues, MSC also have a signicant therapeutic potential for tissue regeneration.35

Cellular senescence has been for long time considered as an exquisitely intracellular event unleashed by activation of

Keywords: senescence; mesenchymal stem cells; IGFBP4; IGFBP7; mass spectrometry; secretome

Abbreviations: CM, conditioned medium; ECM, extracellular matrix; ERK, extracellular signal-regulated kinases; IGF, insulin growth factor; IGFBP4, insulin-like growth factor binding proteins 4; IGFBP7, insulin-like growth factor binding proteins 7; LC-MS, liquid chromatography-mass spectrometry; MSC, mesenchymal stem cells; MUG, 4-methylumbelliferyl-b-D-galactopyranoside; SA-b-gal, senescence-associated b-galactosidase; SASP, senescence-associated secretory phenotype;

SMS, senescence-messaging secretome

Received 11.6.13; revised 31.7.13; accepted 12.9.13; Edited by Y SHi

Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

a cytoplasmic signaling circuitry. Recent studies have evidenced that a pool of molecules secreted by senescent cells, for which the term senescence-associated secretory phenotype (SASP) has been proposed, is associated and may contribute to cellular proliferative arrest through autocrine/paracrine pathways.68 Secreted factors can regulate the senescence response and may represent a danger signal that sensitizes normal neighboring cells to senesce, thus enhancing the ability of damaged cells to enter senescence. In advanced tumor phases, however, cancer cells can take advantage of signaling pathways induced by secreted factors for their growth.6

MSCs benecial inuence in tissue repair has also been attributed to their paracrine activity.9,10 Therefore, changing the pattern of factors secreted by MSC during the senescence process may have a great impact on bodys health. Given the

1Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta, Italy; 2Institute of Biostructures and Bioimaging-IBB, CNR, Napoli, Italy; 3Centro Interuniversitario di Ricerca sui Peptidi Bioattivi-CIRPEB, Napoli, Italy; 4Department of Experimental Medicine, Biotechnology and Molecular Biology Section, Second University of Naples, Napoli, Italy; 5Biomedical Proteomics Research Group, Department of Bioinformatics and Structural Biology, Geneva University, Geneva, Switzerland; 6Institute of Protein Biochemistry and Bioresources-IBP, CNR, Napoli, Italy; 7Institute of Biosciences and Bioresources-IBBR, CNR, Napoli, Italy; 8Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, Temple University, Philadelphia, PA, USA and 9Genome and Stem Cell Center-GENKOK, Erciyes University, Kayseri, Turkey*Corresponding authors: A Chambery, Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, Second University of Naples, Caserta I-81100, Italy. Tel: +39 0823 274535; Fax: +39 0823 274571; E-mail: mailto:[email protected]

Web End [email protected] or U Galderisi, Department of Experimental Medicine, Biotechnology and Molecular Biology Section, Second University of Naples, Napoli I-80138, Italy. Tel: +39 081 5667585; Fax: +39 081 5667547; E-mail: mailto:[email protected]

Web End [email protected]

10These authors contributed equally to this work.

IGFBP4 and IGFBP7 promote premature senescence V Severino et al

paramount importance of autocrine/paracrine signaling in MSC functions, elucidating the composition of the pool of proteins secreted by MSC would enhance our understanding on how these cells affect the environment niche, and support tissue repair, immunomodulation, hematopoietic stem cell self-renew, and tumor growth. To date, studies on the MSC secretome are still in their infancy and mainly refer to the high potential of MSC secretome for the development of therapeutic strategies for the treatment of diseases.11,12

To our knowledge, a comprehensive analysis of the secretome prole of senescent MSC is lacking. In this work, the senescence-inducing properties of conditioned medium (CM) of senescent MSC have been investigated. A bottom-up proteomics approach based on liquid chromatography-mass spectrometry (LC-MS/MS) label-free quantitative analysis has been used to identify and compare the expression levels of secreted proteins in the MSC CM at early and late cultivation phases. Our results provide a detailed characterization of the MSC secretome associated to the senescent phenotype, revealing that selected components of the insulin-like growth factor binding proteins (IGFBPs) family are strongly involved in MSC SASP. We have identied IGFBP4 and IGFBP7 as important senescence-inducing factors, as their single and/or simultaneous immunodepletion abolishes the pro-senescent effect of senescent MSC CM, reduces apoptosis and promotes cell growth. Moreover, the simultaneous addition of rIGFBP4/7 increased senescence and induced apoptosis in MSC. Predicting stem cell fate requires knowledge of how extracellular stimuli are integrated with intracellular signaling networks. Results from an in silico analysis suggest that the extracellular signal-regulated kinases (ERK 1/2) is one of the converging node of the MSC SASP. Accordingly, the induction of MSC senescence program impairs the nuclear/cytosolic localization of active ERK. This study provides an important basis for deciphering the complex extracellular protein networks implicated in MSC cellular senescence and their interplay with the corresponding cytoplasmic signaling circuitry.

Results

CM from senescent MSC triggers senescence in young cells. Senescence of stem cells is caused by a combination of intrinsic irreversible and reversible changes also inuenced by circulating effectors or factors secreted by local stem cell niches.13 Therefore, we decided to investigate the effects of extrinsic signaling on MSC senescence. At rst, properties of young (passage 1, P1) and senescent (passage 10, P10) MSC were evaluated. Following senescence induction, MSC showed a characteristic phenotype including larger and attened cell morphology (Figure 1a). As expected, proliferation rate was signicantly lower in P10 versus P1 cultures (Figure 1b), and this decrease was associated with an increased percentage of senescent cells (Figure 1c). No signicant changes in the apoptotic rate were detected (Figure 1c), conrming the presence of a higher percentage of senescent MSC in P10 compared with P1 cultures.

To determine whether extracellular factors were endowed with senescence-induction properties, proliferation rate,

apoptosis and senescence were evaluated on young MSC cultured with CM from senescent cells (CM-P10). A complementary experiment was performed by culturing senescent cells with CM-P1 from young MSC (Figure 1d). Results demonstrate that CM-P10 was able to trigger senescence in young MSC as suggested by a signicant reduction in their proliferation rate (Figure 1e) and conrmed by 4-methylumbelliferyl-b-D-galactopyranoside (MUG) and senescence-associated b-galactosidase (SA-b-gal) assays (Figures 1g, hj). No modication of the apoptotic rate was detected (Figures 1ik). The specicity of the observed senescence response was attested by the evidence that the incubation of P10 MSC cultures with CM-P1 did not induce any signicant change in proliferation, senescence and apoptosis (Figures 1fk). These results demonstrate that extrinsic factors secreted by senescent MSC are able to promote senescence phenomena in neighboring cells. Once triggered, the senescence process appears to be irreversible, or at least it cannot be reverted by secreted factors produced by young MSC.

Secretome analysis of MSC CM. To identify candidate proteins responsible for the senescence-inducing properties of CM-P10 and to delineate a hallmark of MSC senescent phenotype, a comparative secretome analysis was performed by applying a shotgun proteomics strategy. CM-P1 and CM-P10 were processed for protein precipitation by trichloroacetic acid, and LC-ESI-MS/MS analyses were carried out on peptides deriving from tryptic digestion of proteins. Using high-resolution MS and Mascot database search, 121 proteins were qualitatively identied across both conditions in the MSC secretome. Data were ltered on the basis of probability of protein identication and technical reproducibility, allowing the selection of 66 non-redundant proteins (Supplementary Information, Supplementary Tables S1 and S2). The quantitative analysis performed by spectral counting revealed that 33 proteins were common to both CM-P1 and CM-P10 with no signicant differences in their expression levels (Supplementary Information, Supplementary Table S3). Among differentially expressed proteins, 7 and 13 proteins were uniquely detected in CM-P1 and CM-P10, respectively (Table 1a), whereas a set of proteins was signicantly up- and downregulated in CM-P10 versus CM-P1 (Table 1b).

The identied proteins were analyzed for classical (i.e., signal peptide-driven secretion through the ER/Golgi pathway14) and non-classical (e.g., proteins released through vesicles and extracellular matrix (ECM) proteins15) secretion pathways, revealing that about 94% of the identied proteins were predicted to be secreted. Among them, 57 proteins (B93%) were endowed with the signal peptide for secretion through the ER/Golgi pathway, whereas only four additional proteins (B7%) were predicted to be released by cells through non-classical secretion mechanisms.

A bioinformatic analysis performed by using the Metacore software revealed that a signicant number of differentially regulated proteins was involved in cellmatrix interactions, ECM regulation and protein-folding response (Figure 2a). In detail, the heat map distribution of these proteins show that several upregulated proteins in senescent MSC were involved in cell adhesion, ECM remodeling and connective tissue

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IGFBP4 and IGFBP7 promote premature senescence V Severino et al

Figure 1 CM from senescent MSC triggers senescence in young cells. (a) Induction of replicative senescence was accomplished by repeatedly passaging the cells at P10. Following senescence induction, MSC showed a characteristic phenotype including larger and attened cell morphology with respect to young MSC (P1). (b) Cell proliferation measured by Quick Cell Proliferation Colorimetric Assay Kit II. *Po0.05; **Po0.01 versus P1. (c) Percentage of SA-b-gal-positive cells; **Po0.01 versus P1. Apoptotic cells were detected using uorescein-conjugated Annexin V staining on P1 and P10 MSC. (d) Schematic summary of the experimental workow for the evaluation of the effects of MSC CM on cell proliferation, apoptosis and senescence. (e) Cell proliferation rate evaluated on young MSC cultured with CM-P10 (P1/CM-P10); *Po0.05;

**Po0.01 versus P1 MSC grown in control medium. (f) Cell proliferation rate evaluated on senescent MSC cultured with CM-P1 (P10/CM-P1). (gi) MUG, SA-b-gal and Annexin V assays performed on P1/CM-P10 and on P10/CM-P1; *Po0.05 versus MSC grown in control medium. For all assays, values are means of three independent experiments. (j) Representative microscopic elds of SA-b-gal-positive cells (blue). The mean percentage value of senescent cells (S.D., n 3) is reported in Figure 1h.

(k) Micrographs showing merge of representative elds of P1 and P10 MSC grown under different conditions and stained with Annexin V (green) are showed. Nuclei were counterstained with Hoechst 33342 (blue). The mean percentage value of apoptotic cells (S.D., n 3) is indicated in Figure 1i

degradation (Figure 2b). Several collagen components involved in cartilage development and integrin-mediated cellmatrix adhesion were also upregulated in senescent cells. Furthermore, proteins involved in ER-associated and

cytoplasm protein-folding response were downregulated in senescent MSC (Figure 2b).

Key molecules of the IGF signaling pathway were also differentially regulated in senescent with respect to young

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IGFBP4 and IGFBP7 promote premature senescence

V Severino et al

Table 1 Proteins uniquely (a) and differentially regulated (b) identied in CM-P1 and CM-P10 secretome by high-resolution LC-MS/MS

(a)

ID Accession Protein name Mr (kDa)

No. of peptides

Secretome Pa

Signal Pb

CM-P1 P27797 Calreticulin 48 3 0.37 Y P27658 Collagen alpha-1(VIII) chain 73 2 0.38 Y O43854 EGF-like repeat and discoidin I-like domain-containing protein 3 54 2 0.82 Y P14625 Endoplasmin 92 2 0.50 Y P08238 Heat-shock protein HSP 90-beta 83 4 0.20 N P51884 Lumican 38 8 0.54 Y P23284 Peptidyl-prolyl cis-trans isomerase B 24 2 0.85 N

CM-P10 Q9BRK5 45 kDa calcium-binding protein 42 3 0.59 Y P16112 Aggrecan core protein 250 6 0.57 Y P49747 Cartilage oligomeric matrix protein 83 2 0.27 Y P01034 Cystatin-C 16 2 0.94 Y Q4ZHG4 Fibronectin type III domain-containing protein 1 206 2 0.14 Y P24593 Insulin-like growth factor binding protein 5 31 2 0.92 Y Q9Y287 Integral membrane protein 2B 30 2 0.84 N Q08397 Lysyl oxidase homolog 1 63 3 0.52 Y Q09666 Neuroblast differentiation-associated protein AHNAK 629 2 0.24 N P62937 Peptidyl-prolyl cis-trans isomerase A 18 3 0.34 N P10124 Serglycin 18 2 0.72 Y Q7Z7G0 Target of Nesh-SH3 119 2 0.21 Y P24821 Tenascin 241 2 0.47 Y

(b)

ID Accession Protein name Mr (kDa) No. of peptides

CMP10/ CM-P1

P-value Secretome

Signal Pb

Down P21810 Biglycan 42 6 0.45 0.022 0.71 Y

P12109 Collagen alpha-1(VI) chain 109 11 0.58 0.003 0.23 Y P12110 Collagen alpha-2(VI) chain 109 6 0.23 0.001 0.21 Y P12111 Collagen alpha-3(VI) chain 344 10 0.53 0.006 0.32 Y P35555 Fibrillin-1 312 13 0.56 0.003 0.39 Y Q15063 Periostin 93 8 0.33 0.002 0.27 Y

Up Q99715 Collagen alpha-1(XII) chain 333 52 1.98 0.023 0.33 Y

P08123 Collagen alpha-2(I) chain 129 51 1.80 0.025 0.34 Y P08572 Collagen alpha-2(IV) chain 168 9 2.8 0.034 0.07 Y P21333 Filamin-A 281 15 7.75 1.4E-04 0.45 N P22692 Insulin-like growth factor binding protein 4 28 5 3.63 0.011 0.89 Y Q16270 Insulin-like growth factor binding protein 7 29 9 4.00 0.004 0.54 Y P08670 Vimentin 54 29 2.24 0.013 0.51 N

The Scaffold Software was used to improve protein identication by ltering data following the acceptance criteria of probability greater than 95.0%, as specied by the Protein-Prophet algorithm. The higher number of unique peptides for each protein identication is reported. Mr denotes relative molecular mass

aSecretion prediction according to Secretome P 2.0 server. Proteins with NN-score Z0.5 are predicted as secreted by non-classical secretory pathways

bSecretion prediction according to signal peptide probability of Signal P 4.0 server. Y and N indicate the presence or absence of the signal peptide for secretion

MSC, including several IGFBPs, that are known to have a role in the induction of senescence and cancer.6 In particular, a strong upregulation of IGFBP4 and IGFPB7 was observed in senescent cells, suggesting a role for these factors in triggering senescent phenomena in MSC.

IGFBP4 and IGFBP7 are key factors of senescent MSC CM for triggering senescence phenomena in young MSC. To determine the role of IGFBP4 and IGFBP7 in senescence, cell proliferation, apoptosis and senescence studies were undertaken on young MSC treated with IGFBP4- and/or IGFBP7-immunodepleted CM-P10 (Figure 3a). As, shown, treatment with anti-IGFBP4 and/or anti-IGFBP7 blocked the pro-senescence activity of CM-P10 even at the lowest concentration tested (Figure 3b). It is to be noted that the simultaneous treatment with both anti-IGFBP4 and anti-IGFBP7 provided an enhanced reduction of

senescence induced by CM-P10 on P1 MSC (Figure 3b). Notably, the percentage of senescent cells in P1 cultures incubated with IGFBP4- and IGFBP7-immunodepleted CM-P10 was even lower than control. The analysis of the chromatin status is an additional crucial aspect for an in depth investigation of senescence phenomena. It has been shown that distinct heterochromatin structures accumulate during senescence and could represent a hallmark of this process.16 In P1 cultures incubated with IGFBP4- and/or IGFBP7-immunodepleted CM-P10, a signicant reduction of the HP1 alpha- and H3K9me3-positive cells compared with those supplemented with untreated CM-P10 was observed (Figures 3cf and Supplementary Information, Supplementary Figure S1). This effect was further enhanced by the simultaneous use of both IGFBP4 and IGFBP7 antibodies. In addition, treatment of CM-P10 with both anti-IGFBP4 and anti-IGFBP7 showed a signicant reduction

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IGFBP4 and IGFBP7 promote premature senescence V Severino et al

Figure 2 SASP of senescent MSC. (a) Enriched processes networks for differentially expressed proteins identied by LC-MS/MS analysis. The probability of a random intersection between the differentially expressed proteins with functional processes was evaluated by applying the hypergeometric test by using the Metacore software. (b) Heat map view representing the functional classication of up- and downregulated proteins in CM-P10 versus CM-P1. Signicant functional terms were ranked according to enrichment scores generated using the annotation clustering algorithm in Metacore software

of the apoptosis level (Figure 3g and Supplementary Information, Supplementary Figure S2). Cellular senescence implicates a stable cell growth arrest. To evaluate the effects of IGFBP4/7 soluble factors on MSC growth capability, cell proliferation rate was evaluated on young MSC cultured with CM-P10 alone or treated with anti-IGFBP4 and/or anti-IGFBP7. CM-P10 treatment with anti-IGFBP4 or anti-IGFBP7 showed a signicant increase of cell proliferation rates with respect to P1 MSC cultured with untreated CMP10 at 72 h (Figure 3h). Interestingly, the simultaneous immunodepletion of both IGFBP4 and IGFBP7 resulted in a marked increase of cell proliferation starting from 24 h, thus supporting the view that blocking of IGFBP4/7 signicantly dampens the pro-senescence activity of CM-P10.

The capability of IGFBP4 and IGFBP7 of triggering senescence phenomena in MSC was further conrmed by incubating young MSC with recombinant IGFBP4 and IGFBP7 (rIGFBP4/7). Indeed, the simultaneous addition of both rIGFBP4 and rIGFBP7 increased MSC senescence and induced apoptosis at the highest concentration assayed (Figures 3i and j).

Reversal of the DNA-damage response of young MSC induced by IGFBP4- and IGFBP7-immunodepleted CM-P10. Extensive investigations have revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are linked to cellular senescence and apoptosis.17,18 Therefore, the degree of DNA damage was investigated on young MSC cultured with IGFBP4- and IGFBP7-immunodepleted and non-immunodepleted CM-P10, by determining the level of phosphorylated

histone H2A.X, which is considered a hallmark of damaged DNA nuclear foci.17 A marked reduction of pH2A.X-positive cells was observed when P1 MSC were cultured with CMP10 treated with both IGFBP4 and IGFBP7 antibodies (Figures 4a and b). Moreover, we determined the percentage of 8-oxo-deoxyguanosine (8-oxo-dG), a major product of oxidative damage to DNA.19 In line with our results, the simultaneous blocking of IGFBP4 and IGFBP7 in CM-P10 signicantly decreased the percentage of 8-oxo-dG-positive cells with damaged DNA (Figures 4c and d).

MSC senescence program affects ERK signaling. In response to different signals, multiple transduction pathways and targets are activated to induce cellular senescence responses. To determine the downstream pathway(s) triggered by the MSC senescence-messaging secretome (SMS),6 a network map including the differentially regulated proteins identied by MS analysis was constructed in silico by using the IPA software (Ingenuity Systems Inc., Redwood City, CA, USA) (Figure 5a). As shown, a specic subset of secreted proteins was mapped on a pathway converging, at intracellular level, on ERK 1/2. Previous evidences have demonstrated that the impairment of the nuclear/cytosolic localization of active ERK affects the senescence phenotype in human broblasts.20,21 We therefore supposed that MSC senescence program may involve ERK signaling. To investigate this hypothesis, we analyzed the levels of total and activated ERK (pERK) in young and senescent MSC within different cellular compartments (Figure 5b). The relative distribution was computed as the ratio of the nuclear/ cytoplasmic (N/C) ERK and pERK. Although the ratio N/C for

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IGFBP4 and IGFBP7 promote premature senescence V Severino et al

ERK was constant in P1 and P10, signicant differences were detected with respect to pERK distribution. Indeed, the N/C ratio was strongly reduced only in P10 sample with a

lower relative amount of nuclear versus cytoplasmic pERK in senescent MSC. Furthermore, together with an overall decrease of pERK levels following senescence induction,

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IGFBP4 and IGFBP7 promote premature senescence V Severino et al

a fourfold decrease of nuclear pERK was revealed in P10 versus P1 MSC.

Discussion

Senescence is a physiological phenomenon leading to permanent cell growth arrest and impairment of cellular functions.1,22 The extracellular microenvironment has a key role in regulating and determining the cellular response to senescence signaling. It is known that senescent cells secrete a unique repertoire of molecules (SASP) for which the term SMS has been recently proposed.6 Although the importance of changes in SMS has long been recognized, the molecular

details of senescence signaling remain elusive. Furthermore, to the best of our knowledge, the senescence-induced extracellular response of stem cells has not been yet investigated. Given the peculiar properties of stem cells, including their multilineage differentiation capability, it is of fundamental importance to understand the response of stem cells to senescence signaling, also in view of their high potential in regenerative medicine and cancer therapy.

We demonstrate that MSC SASP implements a full senescence response in young cells, suggesting the presence, in the pool of secreted molecules, of factors capable of triggering, alone and/or in combination, senescence mechanisms through autocrine/paracrine signaling

Figure 4 Reversal of the DNA-damage response of young MSC induced by CM-P10 by IGFBP4 and IGFBP7 blocking. (a and b) Phosphorylated H2A.X staining performed on young MSC cultured with untreated and IGFBP4 and/or IGFBP7 antibody-treated CM-P10; *Po0.05; **Po0.01 versus P1 MSC grown with untreated CM-P10.

Fluorescence micrographs showing merge of representative elds of cells stained with anti-pH2A.X (green) and Hoechst 33342 (blue). (c and d) 8-oxo-dG staining performed on young MSC cultured with untreated and antibody-treated CM-P10. *Po0.05, **Po0.01 versus P1 MSC grown with untreated CM-P10. Fluorescence micrographs showing merge of representative elds of cells stained with anti-8-oxo-dG (green). Nuclei were counterstained with Hoechst 33342 (blue)

Figure 3 Secreted IGFBP4 and IGFBP7 induce senescence in young MSC. (a) Schematic summary of the experimental workow for the investigation of the senescence-induction potential of IGFBP4 and IGFBP7. (b) MUG assay performed on young MSC cultured with untreated and antibody-treated CM-P10 at different concentrations (3, 9 and 20 mg/ml). Values are means of three independent experiments; *Po0.05; **Po0.01 versus P1 MSC grown with untreated CM-P10. (c and d) HP1 alpha detection performed on young MSC cultured with untreated and antibody-treated CM-P10. Micrographs showing merge of representative elds of P1 MSC grown under different conditions and stained with anti-HP1 alpha (green) to identify nuclear heterochromatin are showed. Nuclei were counterstained with Hoechst 33342 (blue); *Po0.05;

**Po0.01 versus P1 MSC grown with untreated CM-P10. (e and f) H3K9me3 detection performed on young MSC cultured with untreated and antibody-treated CM-P10. Micrographs showing merge of representative elds of P1 MSC grown under different conditions and stained with anti-H3K9me3 (green) are showed. Nuclei were counterstained with Hoechst 33342 (blue); *Po0.05; **Po0.01 versus P1 MSC grown with untreated CM-P10. (g) Apoptosis assay of young MSC cultured with untreated and antibody-treated CM-P10; *Po0.05; versus P1 MSC grown with untreated CM-P10. (h) Cell proliferation assay on young MSC cultured with untreated and antibody-treated CM-P10. *Po0.05; **Po0.01 versus P1 MSC grown with untreated CM-P10. (i and j) MUG and Annexin V assays performed on young MSC cultured with rIGFBP4 and/or rIGFBP7 at different concentrations (0.5, 2.5 and 10 mg/ml). Values are means of three independent experiments; *Po0.05; **Po0.01 versus P1 MSC grown in control medium

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IGFBP4 and IGFBP7 promote premature senescence V Severino et al

Figure 5 MSC senescence program involves the ERK 1/2 signaling. (a) Protein interaction network including a subset of differentially expressed proteins secreted by senescent MSC. The core molecules of the network were found to converge at extracellular level on IGF and TGF beta and, at intracellular level, on ERK 1/2 signaling pathways. According to IPA categorization, proteins uniquely detected or upregulated in senescent MSC are colored in orange and red, respectively; proteins uniquely detected or upregulated in young MSC are colored in blue and green, respectively. The pathway components identied by the algorithm or with no signicant differences in their expression levels are reported in gray. Molecules are named according to IPA software as follows: COL12A1, Collagen alpha-1(XII); COL8A1, Collagen alpha-1(VIII); COL6A1, Collagen alpha-1(VI); COL6A3, Collagen alpha-3(VI); POSTN, Periostin; IGFBP, insulin-like growth factor binding protein; IGF, insuline-like growth factor; COL6A2, Collagen alpha-2(VI); COL1 A2, Collagen alpha-2(I); TGF beta, transforming growth factor beta; Mmp, Metalloproteinase protein family; COMP, Cartilage oligomeric matrix protein; TNC, Tenascin; ACAN, Aggrecan core protein; FBN1, Fibrillin-1; LUM, Lumican; BGN, Biglycan. (b) Western blot for ERK and pERK in N/C fractions of P1 and P10 MSC. To assess the purity of the N/C fractions, immunoblot analysis was performed with anti-GAPDH, as cytoplasmic marker, and anti-H1.2, as nuclear marker. Relative density of the bands was normalized to b-actin. (c) Schematic summary of the active ERK nucleo-cytoplasmic trafcking in the MSC response to replicative senescence induction

cascades. By performing a secretome-wide proling of young and senescent MSC by LC-MS/MS, we have identied a subset of proteins characterizing their senescent phenotype, several of which involved in ECM remodeling and unfolded protein response. Consistent with our ndings are previous studies reporting that ECM components can also inuence their environment by mediating chemical and mechanical cues for tissue morphogenesis, proliferation and differentiation.23 Upon cellular aging and senescence, an overall loss of junctional integrity, a thinning of the basement membrane and a progressive loss of ECM architecture are observed following a dysregulation in synthesizing the correct matrix components.24,25 Our results show that besides the occurrence of signicant changes in collagen composition, other ECM proteins (e.g., Fibrillin-1, Periostin, Lumican, Tenascin and Biglycan), involved in the maintaining of the structural and functional integrity of ECM, are differentially regulated in senescent cells. Furthermore, two components of the cartilage ECM matrix (i.e., Aggrecan and Cartilage oligomeric matrix protein, COMP) are specically detected in senescent MSC. It has been reported that COMP interacts and is covalently cross-linked to the aggrecan core protein, and that the amount of these complexes increases with age.26

In addition to ECM components, proteins associated to unfolded protein response are downregulated in senescent MSC. This nding is in agreement with the expected attenuation of molecular chaperone function and the resulting increase of protein misfolding and aggregation occurring during aging and senescence.27 Among them, heat-shock protein 90-beta, downregulated in senescent MSC, is

reported to be required for the correct assembly and function of telomerase complexes, which is an essential enzyme regulating cell life span.28

Key regulators of the IGF signaling pathways are differentially expressed in senescent versus young MSC. In particular, we detect several members of the IGFBP family(i.e., IGFBP2, IGFBP3, IGFBP4, IGFBP5 and IGFBP7). Although a role in senescence has been previously reported for IGFBP3 in human endothelial cells29 and MCF-7 breast cancer cells,30 we did not detect signicant expression changes for this factor and his upstream regulator plasminogen activator inhibitor 1. This result, together with the simultaneous detection of other differentially expressed IGFBPs, suggests the activation of alternative senescence-inducing pathways in MSC. In particular, IGFBP5, previously reported to trigger senescence through a p-53 dependent mechanism,31,32 is uniquely detected in senescent MSC.

The strong upregulation of two additional IGFBPs(i.e., IGFBP4 and IGFBP7) in senescent MSC prompted us to investigate their involvement in triggering MSC senescence and results provide new insights on the capability of IGFBP4 and IGFBP7 in promoting senescence in MSC. We indeed nd that IGFBP4 and IGFBP7 are key players in MSC SASP, because are involved in the induction of a senescent effect that strongly inuences the functions of young cells. Blocking of IGFBP4 and/or IGFBP7 in CM-P10 reduces their prosenescent effect on P1 MSC cultures. It is noteworthy that the simultaneous inhibition of IGFBP4 and IGFBP7 suppresses apoptosis and promotes cell growth, supporting their pleio-tropic effect on MSC biology. Furthermore, the simultaneous

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IGFBP4 and IGFBP7 promote premature senescence V Severino et al

addition of rIGFBP4/7 increased senescence and induced apoptosis in MSC, and these effects were also observed with single addition of rIGFBP7. Our ndings are in agreement with recent studies, demonstrating that IGFBPs have a causal role in the induction of senescence in cancer systems.6 Reportedly, overexpression of IGFBP7 triggers BRAFV600E-induced senescence in human melanocytes33,34 and is considered a

tumor suppressor, as it reduces breast tumor growth by induction of senescence and apoptosis pathways.35 Similarly, expression levels of IGFBP4 mRNA are increased in senescent hepatic stellate cells36 and following induction of senescence-associated genes by 5-bromodeoxyuridine in HeLa cells.37

A critical aspect of senescence signaling is to unravel the connection between the SMS and the downstream effectors and pathways.6 Recent evidences support the hypothesis that SMS-associated factors may be involved in distinct but integrated signaling pathways, and that the dynamic activation of intracellular cascades is responsible for the cell decision to eventually engage a senescence program.6 However, the intracellular targets of SMS factors secreted by a given cell type have not been fully elucidated.6 By using a bioinformatic approach, a specic subset of proteins of MSC SASP was mapped on a pathway converging on ERK 1/2. Wajapeyee et al.33,38 demonstrated that IGFBP7 suppresses the phosphorylation of ERK and mediates its antiproliferative effects through a negative feedback signaling in oncogenic BRAFV600E-mediated senescence. In sharp contrast, previous evidences suggested that the expression of BRAFV600E induces the activation of ERK 1/2. These studies clearly demonstrate an involvement of the ERK signaling in the oncogene-induced senescence and point out the crucial role of ERK signaling in mediating different antiproliferative events such as apoptosis, autophagy and senescence.20

The plethora of signals that activate the ERK pathway and its numerous substrates suggest that the ne control of the spatiotemporal regulation of ERK activity is critical for determining opposite cell fates.20 Activation of cytoplasmatic ERK 1/2 through its phosphorylation leads to nuclear translocation thus allowing the interaction with specic nuclear substrates and the induction of specic programs of gene expression. The regulation of ERK nucleo-cytoplasmic trafcking modulates the transfer efciency of the signaling carried by activated ERK to the cell.20 Accumulating evidences show that nuclear relocalization of active ERK is impaired in senescent human broblasts, and that this dysregulation may account for the irreversible proliferative arrest of senescent cells.20 The

impaired nuclear localization of active ERK and the loss of proliferative potential have been proposed to originate from either impaired nuclear import or from ERK inactivation. This interpretation is supported by the high activity of the nuclear ERK phosphatase MKP2/DUSP4 in senescent cells.21

Our results also attest a dysregulation of active ERK localization in stem cell senescence program, as a lower amount of nuclear versus cytoplasmic pERK is revealed in senescent MSC together with a decrease of nuclear pERK in P10 versus P1 MSC (Figure 5c). This nding strongly supports previous studies and further delineates an unexpected complexity of ERK signaling mechanisms in cellular senescence.20 Indeed, we might envisage a scenario in which the nuclear/cytosolic localization of active ERK may

be related to the MSC senescent phenotype as previously demonstrated for human broblasts.

Although more extensive studies are needed to expand our knowledge on the mechanisms underpinning the impaired ERK nucleo-cytoplasmic trafcking in stem cells, we also believe that our results pave the way to further investigations aiming to modify, in the near future, the current in vitro MSC expansion protocols for therapeutic purposes, thereby preventing or reducing the occurrence of negative senescence-related effects, and to better understand the complex process of senescence and aging in stem cells.

Materials and MethodsMSC cultures and preparation of CM. MSC were obtained and cultured as previously described.39 Bone marrow was obtained from healthy donors after informed consent. Cells were separated on Ficoll density gradient (GE Healthcare, Milan, Italy) and the mononuclear cell fraction was collected and washed in PBS. Cells (12.5 105/cm2) were seeded in alpha-MEM containing 10% FBS and

bFGF. After 72 h, non-adherent cells were discarded and adherent cells were further cultivated to conuency and amplied at P1. Replicative senescence was induced by repeatedly passaging the cells at P10. P1 and P10 cultures are referred to as young and senescent MSC, respectively. In our experimental conditions, it was veried that MSC cultures fullled the minimal criteria to dene MSC.40 All cell culture reagents were obtained by Euroclone Life Sciences (Milan, Italy) and Hyclone (Logan, UT, USA).

To harvest MSC secretome, 80% conuent P1 and P10 MSC cultures were extensively washed with PBS and transferred to a chemically dened, serum-free culture medium for an overnight incubation. Then, the CM from young (CM-P1) and senescent (CM-P10) MSC were collected and used at 50% in fresh alpha-MEM for in vitro cultivation of senescent and young MSC, respectively. Three independent culture preparations were prepared and pooled to address biological variation.

CM preparation for LC-MS/MS analysis. CM-P1 and CM-P10 were claried by centrifugation, lyophilized and resuspended in 1 ml of 50 mM NH4HCO3. The proteins were then precipitated with 20% trichloroacetic acid for 30 min on ice and centrifuged for 15 min at 17 500 g.4143 Pellets washed with

diethyl ether and acetone were air dried at room temperature, resuspended in 50 mM NH4HCO3, pooled and thoroughly sonicated into an ultrasonic bath for 1015 min. Proteins were reduced with 2.5 mM dithiothreitol (nal concentration) at 60 1C for 30 min and carbamidomethylated with 7.5 mM iodoacetamide (nal concentration) at room temperature in the dark for 30 min. Enzymatic hydrolysis was performed by the addition of 10 ml of tosyl phenylalanyl chloromethyl ketone-treated trypsin solution (5 ng/ml) to the reduced and alkylated mixture. Digestion was performed by incubation at 37 1C for 16 h, followed by a second addition of 10 ml of trypsin incubated at 37 1C for 3 h. After digestion, samples were dried under vacuum in a SpeedVac Vacuum (Savant Instruments,

Holbrook, NY, USA), resuspended in H2O/CH3CN/formic acid (FA) 95%/5%/0.1%, and peptides were puried by using macro spin columns (Harvard Apparatus, Holliston, MA, USA). Samples were dried, resuspended in 120 ml of 0.1% FA and centrifuged at 17 500 g for 15 min. Aliquots of the supernatant (6 ml) were

analyzed by ESI LTQ-OT MS.4143

LC-MS conguration and protein identication. ESI LTQ-OT MS was performed on a LTQ Orbitrap Velos from Thermo Electron (San Jose, CA, USA) equipped with a NanoAcquity system from Waters (Waters Corporation, Manchester, UK).43 Peptides were trapped on a home-made 5-mm 200 Magic

C18 AQ (Michrom, Auburn, CA, USA) 0.1 20 mm pre-column and separated on

a home-made 5-mm 100 Magic C18 AQ (Michrom) 0.75 150 mm column with

a gravity-pulled emitter. The analytical separation was run for 65 min, using a gradient of H2O/FA 99.9%/0.1% (solvent A) and CH3CN/FA 99.9%/0.1%

(solvent B), at a ow rate of 220 nl/min as follows: 5% solvent B for 1 min, from 5 to 35% solvent B in 54 min and from 35 to 80% solvent B in 10 min. For MS survey scans, the Orbitrap resolution was set to 60 000 and the ion population was set to 5 105 with an m/z window from 400 to 2000. Five precursor ions were

selected for collision-induced dissociation in the LTQ by setting the ion population

to 7 103 (isolation width of 2 m/z). The normalized collision energies were set to

35% for collision-induced dissociation.

Cell Death and Disease

IGFBP4 and IGFBP7 promote premature senescence V Severino et al

Data processing was performed as previously described.44,45 The Mascot

software (Matrix Science, London, UK; version 2.2.0) was used for database searching against the Uniprot_sprot database release 2011_09 (21 September 2011, selected for Homo sapiens, 20 323 entries) assuming trypsin as digestion enzyme. Mascot was searched with a fragment ion mass tolerance of 0.60 Da and a parent ion tolerance of 25 p.p.m. Iodoacetamide derivative of cysteine was specied in Mascot as a xed modication. Oxidation of methionine was specied in Mascot as a variable modication.

Scaffold software (version 4.0.1, Proteome Software Inc., Portland, OR, USA) was used to validate MS/MS-based peptide and protein identications. Peptide identications were accepted if they could be established at 495% probability.46

Protein identications were accepted if they could be established at Z95% probability and contained at least two identied unique peptides across replicate injection for each condition. Protein probabilities were assigned by the ProteinProphet algorithm.47 Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony. A false discovery rate value of 0.1% was calculated by using the Scaffold software for MS/MS validation.

Quantitative analysis. For protein quantication, for each identied protein, the number of spectral counts (the number of MS/MS spectra associated with an identied protein) was used to identify differentially expressed proteins.48,49 To this

end, normalized spectral counts were calculated by dividing the spectral counts for an identied protein by the sum of the spectral counts per sample. The statistical evaluation of proteins differentially expressed between the CM-P1 and CM-P10 samples was performed by applying the Fishers exact test (Po0.05).

Bioinformatics analyses. Secreted and differentially expressed proteins identied in CM-P1 and CM-P10 were imported into the Ingenuity Pathways Analysis (IPA, Ingenuity Systems Inc.) or Metacore (GeneGo, St. Joseph, MI, USA) software for batch analysis and identication of the canonical pathways as previously reported.41,43,50 An assessment of signicantly enriched processes

networks for differentially expressed proteins was performed by evaluating the probability of a random intersection between the differentially expressed proteins with functional processes by applying the hypergeometric test.50 The heat map representing the functional classication of up- and downregulated proteins in CM-P10 versus CM-P1 was constructed by clustering signicant functional terms according to the Metacore annotation algorithm by using the TIBCO Spotre software (Somerville, MA, USA). Proteins with a predicted N-terminal signal sequence were identied by using SignalP 4.0,51 available at http://www.cbs.dtu.dk/services/SignalP/

Web End =http://www.cbs.dtu.dk/services/SignalP/ (set for eukaryotes using both neural networks and hidden Markov models), and were considered to be secreted via a classical pathway (endoplasmic reticulum/Golgi-dependent pathway). Identied proteins were also analyzed for secretion pathways according to SecretomeP 2.0 Server (http://www.cbs.dtu.dk/ services/SecretomeP/

Web End =http://www.cbs.dtu.dk/ services/SecretomeP/ ).52 If the neural network exceeded or was equal to a value of 0.5 (NN-score Z0.50), but no signal peptide was predicted, proteins were considered potentially secreted via a non-classical pathway.

Immunodepletion experiments. IGFBP4 and/or IGFBP7 were immuno-depleted in CM-P10 by incubating CM and cells with a polyclonal anti-human IGFBP4 (Upstate Biotechnology, Lake Placid, NY, USA; catalog no. 06-109) and a polyclonal anti-human IGFBP7 (Santa Cruz Biotechnology, Dallas, TX, USA; catalog no. sc363293) at a nal concentration of 3, 9 and 20 mg/ml for 30 min at 4 1C. Then, P1 MSC were cultured for 24 h at 37 1C with the CM-P10 treated with antibodies. After the incubation period, senescence was evaluated by MUG assay as described below. Cell proliferation and apoptosis were assessed by using CM-P10 immunodepleted with the highest antibody concentration (20 mg/ml).

Effect of recombinant IGFBP4 and IGFBP7 on MSC senescence and apoptosis. Recombinant human IGFBP4 and/or IGFBP7 (rIGFBP4 and rIGFBP7; PeproTech, London, UK; catalog nos. 350-05B and 350-09, respectively) were added to young MSC (P1) cultures at a nal concentration of0.5, 2.5 and 10 mg/ml. After a 36-h incubation period, senescence and apoptosis were evaluated by MUG and annexin V assays, respectively, as described below.

Cell proliferation and annexin V assays. Cell proliferation was evaluated by Quick Cell Proliferation Assay kit II (Biovision, Milpitas, CA, USA). One thousand cells were seeded in 96-well culture plates. At 1, 2 and 5 days post plating, cells were collected and counted. The ratio of the total number of cells at

day n to the number of cells at day n-1 was regarded as the cell proliferation rate.53 Apoptotic cells were detected using uorescein-conjugated annexin V (Roche, Milan, Italy) following the manufacturers instructions.

Senescence assays. The percentage of senescent cells was calculated in situ by the number of blue b-galactosidase-positive cells out of at least 500 cells in different microscope elds as already reported.54 For the quantitative SA-b-gal assay, 4-MUG was used as substrate of b-galactosidase. 4-MUG does not uoresce until cleaved by the enzyme to generate the uorophore 4-methylumbelliferone. Assay was carried out on lysates obtained from cells that were grown in 96-wells plates as reported.55 The production of the uorophore was monitored at an emission/excitation wavelength of 365/460 nm.

Cell fractionation. Cell fractionation was performed essentially as previously described.56 P1 and P10 MSC were lysed in 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.5 mM spermidine and 0.5% Nonidet P40. Following an incubation of 5 min at 4 1C, nuclear and cytosolic fractions were obtained after centrifugation for 5 min at 500 g. The nuclei were washed three times in PBS, resuspended in

the lysis buffer and further centrifuged at 17 500 g.

Immunocytochemistry. Mouse monoclonal anti-8-oxo-dG (clone 2E2; Trevigen, Gaithersburg, MD, USA), rabbit monoclonal anti-phospho histone H2A.X (pH2A.X), rabbit polyclonal anti-HP1alpha (Cell Signaling, Danvers, MA, USA) and rabbit polyclonal anti-histone H3-trimethyl K9 (anti-H3K9me3, Abcam, Cambridge, UK) antibodies were used for the detection of 8-oxoguanine, pH2A.X, HP1 alpha and H3K9me3, respectively, according to the manufacturers protocol. Nuclei were counterstained with Hoechst 33342 and cells were observed through a uorescence microscope (Leica, Milan, Italy). The percentage of 8-oxo-dG-, HP1 alpha-, pH2A.X- and H3K9me3-positive cells was calculated by counting at least 500 cells in different microscope elds.

Western blot analysis. For western blot analysis, proteins from P1 and P10 MSC nuclear and cytosolic fractions (15 mg) were resolved by SDS-PAGE on a 415% Mini-Protean TGX precast gel (Bio-Rad, Milan, Italy) under reducing conditions and transferred onto nitrocellulose membrane (Bio-Rad) with a Trans-Blot Turbo electroblot apparatus (Bio-Rad). For the assessment of the purity of the nuclear and cytoplasmic fractions, monoclonal anti-GAPDH (1:20 000, Sigma, Milan, Italy; catalog no. G8795) and polyclonal anti-H1.2 (1:1000, Abcam; catalog no. ab17677) were used as cytoplasmic and nuclear markers, respectively. For the detection of ERK 1/2, anti-ERK 1 was used at a dilution of 1:2000 (Santa Cruz Biotechnology; catalog no. sc-93). Active (pERK) was detected by using an anti-phospho ERK 1/2 diluted 1:1000 (Cell Signaling; catalog no. 9101 L). Immunoreactive protein bands were visualized by the enhanced chemiluminescence (ECL) Plus Western Blotting Detection System (GE Healthcare) according to the manufacturers instructions. Western blot results were normalized by stripping and reprobing the membranes with anti-b-actin monoclonal antibody (Sigma-Aldrich, Milan, Italy; catalog no. A2228) used for blot normalization.

Densitometry analysis was performed with ImageJ software (http://rsb.info.nih.gov/ij/

Web End =http://rsb.info.nih.gov/ij/).

Conict of InterestThe authors declare no conict of interest.

Acknowledgements. This work was partially supported by Ministry of Foreign Affairs Republic of Italy (Senescence of stem cells and Rett syndrome), by European Energy Atomic Community FP-7-Fission 2012 RISK-IR) to UG and by Progetto PON - Ricerca e Competitivit 20072013 - PON01_00802 entitled: Sviluppo di molecole capaci di modulare vie metaboliche intracellulari redoxsensibili per la prevenzione e la cura di patologie infettive, tumorali, neurodegenerative e loro delivery mediante piattaforme nano tecnologiche.

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Title

Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

Author

Severino, V; Alessio, N; Farina, A; Sandomenico, A; Cipollaro, M; Peluso, G; Galderisi, U; Chambery, A

Pages

e911

Publication year

2013

Publication date

Nov 2013

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Springer Nature B.V.

e-ISSN

20414889

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Scholarly Journal

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English

DOI

https://doi.org/10.1038/cddis.2013.445

ProQuest document ID

1786138491

Insulin-like growth factor binding proteins 4 and 7 released by senescent cells promote premature senescence in mesenchymal stem cells

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