Clusterin silencing restores myoblasts viability and down modulates the inflammatory process in osteoporotic disease
Targeting new molecular pathways leading to Osteoporosis (OP) and Osteoarthritis (OA) is a hot topic for drug discovery. Clusterin (CLU) is a glycoprotein involved in inflammation, proliferation, cell death, neoplastic disease, Alzheimer disease and aging. The present study focuses on the expression and the role of CLU in influencing the decrease of muscle mass and fiber senescence in OP-OA condition.
Vastus lateralis muscle biopsies were collected from 20 women with OP undergoing surgery for fragility hip fracture and 20 women undergoing arthroplasty for hip osteoarthritis.
We found an overexpression of CLU in degenerated fibers in OP closely correlated with interleukin 6 (IL6) and histone H4 acetylation level. Conversely, in OA muscle tissues we observed a weak expression of CLU but no nuclear histone H4 acetylation. Ex vivo studies on isolated human myoblasts confirmed CLU overexpression in OP as compared to OA (p < 0.001). CLU treatment of isolated OP and OA myoblasts showed: modulation of proliferation, morphological changes, increase of histone H4 acetylation and induction of myogenin (MYOG) activation in OP myoblast only. In OP condition, functional knockdown of CLU by siRNA restores proliferative myoblasts capability and tissue damage repair, carried out by an evident upregulation of Transglutaminase 2 (TGM2). We also observed downmodulation of CX3CR1 expression with consequent impairing of the inflammatory infiltrate recruitment.
Results obtained suggest a potential role of CLU in OP by influencing myoblasts terminal differentiation, epigenetic regulation of muscle cell differentiation and senescence. Moreover, CLU silencing points out its role in the modulation of tissue damage repair and inflammation, proposing it as a new diagnostic marker for muscle degeneration and a potential target for specific therapeutic intervention in OP related sarcopenia.
KeywordsClusterin (CLU) Osteoporosis Osteoarthritis Sarcopenia Muscle waist Osteoporosis marker
bone mineral density
body mass index
Harris hip score
joint space narrowing
nuclear factor kappa-light-chain-enhancer of activated B cells
CX3C chemokine receptor 1
Osteoporosis (OP), Osteoarthritis (OA) and sarcopenia are multifactorial diseases with a clear genetic component, in which both immune response and chronic inflammation play an important role. The pathogenic process of these degenerative diseases leads, eventually, to the degeneration of cartilage, subchondral bone-to-bone abnormalities and severe impairment of joint function and, in the case of OP, also to sarcopenia. In particular, sarcopenia is an aging-induced generalized pathological condition characterized by loss of muscle mass and age-related functions [1, 2]. The conditions leading to muscle loss involve different intracellular signaling pathways, apoptosis, mitochondrial dysfunctions  and the alteration of lipidic pathways. The development and homeostasis of different systems such as the nervous, muscular and bone relies on the presence of key regulatory molecules like growth factors, soluble mediators and their respective receptors. A crucial role in aging is played by proinflammatory factors such as interleukin 6 (IL6) that mediates cell–cell interactions. Aging and the subsequent atrophy of muscle fibers lead to a reduction of mechanical bone stimuli exercised through the tendons, which regulate bone development and remodelling, thus bringing to a degenerative process such as OA and OP . In this context, Clusterin (CLU) is a new pleiotropic factor potentially involved in the stimulation of inflammatory cytokines such as IL6 and lipid metabolism, cell differentiation, tissue remodeling and neoplastic diseases [5, 6, 7].
Emerging evidences suggest that CLU plays an important role in muscle and bone homeostasis. CLU is an heterodimeric disulfide-linked protein of ~ 75–80 kDa involved in several physiological processes including lipid transportation in serum, cellular senescence, aging and various age-related diseases, like neurodegeneration, inflammation, vascular damage, diabetes and tumourigenesis [8, 9, 10, 11, 12, 13, 14, 15, 16, 17]. Two different CLU transcripts, derived from alternative splicing, have been identified: one coding for a nuclear form (nCLU, 50–55 kDa) and a second one coding for a highly glicosilated cytoplasmatic form (sCLU, 40 kDa) found also in biological fluid. In particular sCLU is an intra- and extracellular chaperon stress inducible protein and it has also been functionally implicated in signalling pathways that regulate development, differentiation and apoptotic cell death. CLU polymorphisms were recently found to be associated with Alzheimer’s disease [18, 19, 20]. Existing studies referring to sCLU in degenerative joint diseases are limited and its role in these pathologies is still obscure. The main purpose of the present study was therefore to characterize new pathways and molecular targets involved in the onset and progression of OP and OA. We focused on the role of CLU, which is involved also in calcium metabolism , to clarify its function in these pathologies and its potential action in reduction of muscular mass leading to sarcopenia and its possible involvement in the inflammatory process that characterizes OP and OA condition . Through the in vitro experiments, we analyzed the effect of CLU on proliferation, on DNA acetylation levels and on myogenin (MYOG) activation, the main marker of myoblasts differentiation. Furthermore, we characterized the effect of CLU silencing by siRNA on: proliferation, expression of genes and proteins involved in tissue damage repair and in the initiation of the inflammatory process.
Main characteristics of OP and OA patients
80.58 ± 6.3 years
67.63 ± 8 years
24.56 ± 4.6
27.32 ± 4.4
42.89 ± 1.3
43.68 ± 1.4
− 2.8 ± 0.7
− 0.75 ± 0.5
T-score (femoral neck)
− 3 ± 0.6
− 0.67 ± 0.11
47.26 ± 5
Bone mineral density evaluation (DXA)
DXA was performed with a Lunar DXA apparatus (GE Healthcare, Madison, WI, USA). Lumbar spine (L1–L4) and femoral (neck and total) scans were performed, and bone mineral density (BMD) was measured according to manufacture’s recommendations . Dual-energy X-ray absorptiometry measures BMD (in grams per square centimeter), with a coefficient of variation of 0.7%. For patients with fragility fractures, BMD was measured on the uninjured limb. For OA patients, measurements were performed on the non-dominant side, with the participants supine on an examination table with their limbs slightly abducted . DXA exam was performed 1 day before surgery for OA patients, and 1 month after surgery for OP patients. The results were expressed as T-scores.
Harris hip score (HHS)
The Harris hip score (HHS) was measured to evaluate the level of joint dysfunctionality in OA patients. It includes 4 sections. Pain—scoring between 0 and 44 points. Function—up to 47 points divided into walking functions (up to 33 points), and daily activities (up to 14 points). Absence of deformity 4 points, and movement range 5 points .
Hip x-rays were performed in order to check the fracture or to assessed hip OA. Kellgren-Lawrence scale was used in order to determine the severity of OA. The Kellgren and Lawrence system is a method of classifying the severity of OA using five grades. This classification was proposed by Kellgren et al.  in 1957. It includes: grade 0 if no radiographic features of OA are present; grade 1 if doubtful joint space narrowing (JSN) and possible osteophytic lipping; grade 2 if definite osteophytes and possible JSN on anteroposterior weight-bearing radiograph; grade 3 if multiple osteophytes, definite JSN, sclerosis, possible bony deformity; grade 4 if large osteophytes, marked JSN, severe sclerosis and definite bony deformity. Two orthopaedists independently assessed all radiographs. Patients with a grade of K−L ≥ 2 were considered osteoarthritic.
During open surgery for hip arthroplasty, muscle biopsies were taken from the upper portion of the vastus lateralis. Sample withdrawals were performed for histological analysis excluding macroscopic alteration of skeletal muscle biopsy as necrosis areas.
Muscle biopsies were fixed in 4% paraformaldehyde for 24 h and paraffin embedded. 3 μm sections were stained with hematoxylin and eosin (H&E) and subsequently the histomorphometric evaluations were carried out independently by two pathologists. Specifically, both the diameter of 200 muscle fibers and the number of fibers per μm2 were measured for each sample by using digital image (iScan Coreo—ImageView software, Ventana, Roche, USA). In order to assess fibers atrophy, a minimum of 200 muscle fibers per biopsy have been evaluated, comparing minimum transverse diameter and cross-sectional area of type I and type II fibers for relative prevalence. A threshold diameter lower than 30 μm characterized atrophic fibers.
Primary antibodies used for immunohistochemistry (IHC) analysis
Mouse monoclonal, R&D Systems
Goat polyclonal, Santa Cruz Biotechnology
Anti-acetyl histone H4
Rabbit polyclonal, Upstate
Mouse monoclonal, Sigma-Aldrich
Cell culture and CLU conditioning
Human myoblast cells were extracted ex vivo and grown on gelatin matrix (Fluka) in complete growth medium supplied with 15% FBS, insulin 1 mg/ml, FGF 5 μg/ml and EGF 10 μg/ml. For CLU treatment, myoblasts were seeded in 96 well multi-wells at 8000 cell/cm2, in 6 well multi-wells at 9000 cell/cm2 and in 4 well Lab. Tek II Chamber slides at 8000 cell/cm2, in complete growth medium (F14 + 15% FBS). After an overnight culture, medium was removed and human recombinant CLU (Endogen) was added at a final concentration of 2 μg/ml for 6 days. Untreated cells were used as control. After 3 and 6 days from treatment cells were counted through trypan blue method. After 48 h, cells were collected for RNA extraction and after 72 h cells were fixed in formalin 10% for ICC analysis.
In this study, OP and OA myoblasts were silenced for CLU gene. Double-strand purified RNAs pre-designed from Sigma-Aldrich (Milan, Italy) with specific sequences for CLU gene were used: sense 5′-GGAUGAAGGACCAGUGUGAdTdT-3′ and antisense 5′-UCACACUGGUCCUUCAUCCdTdT-3′. Non-specific sequences were used as transfection control (scramble): sense 5′-UUCUCCGAACGUGUCACGUdTdT-3′ and antisense 5′- ACGUGACACGUUCGGAGAAdTdT-3. Cells were seeded in 96 well multi-wells at 8000 cell/cm2, in 6 well multi-wells at 9000 cell/cm2 and in 4 well Lab. Tek II Chamber slides at 8000 cell/cm2, in complete growth medium (F14 + 15% FBS); after an overnight culture, when the cell confluence was at 60–70%, it proceeded with transfection through Lipofectamine® 2000 Transfection Reagent (Invitrogen, Thermo Fisher Scientific), following the guidelines indicated by the company. Lipofectamine and siRNAs (33 nM final concentration) were diluted in Opti-MEM (Gibco-BRL) and incubated for 20 min at room temperature before being added to cells. At the end of the incubation (6 h), transfection complex was removed and complete growth medium was added to cells. After 24, 48 and 72 h cells were counted through trypan blue method. After 48 h from transfection, cells were collected for RNA extraction and after 72 h cells were fixed in formalin 10% for ICC analysis. The efficiency of silencing was confirmed through RT-PCR (Fig. 5).
RNA extraction, RT-PCR and qRT-PCR
Sequences of primers used for RT-PCR and qRT-PCR
T annealing (°C)
Sense: 5′-GAGGAGCTGGTCTTAGAGAGG-3 ́
Antisense: 5′-CGGTCACGACACTGAAGGTG-3 ́
Primary antibodies used for immunocytochemistry (IHC) analysis
Goat polyclonal, Santa Cruz Biotechnology
Mouse monoclonal, Santa Cruz Biotechnology
Anti-acetyl histone H4
Rabbit polyclonal, Upstate
Rabbit polyclonal, Mo Bi Tec Molecular Biologische Technologie
Rabbit monoclonal, AbCam
All values provided in the text and figures are means of three independent experiments ± standard deviations (SD). The positive stain in IHC and ICC was evaluated by two independent observers. Unpaired t-tests were performed to assess inter-group statistical differences. Differences were considered statistically significant for p < 0.05.
The OP group included 20 patients with fragility hip fracture, T-score ≤ − 2.5 SD and K–L score from 0 to 1. The OA group included 20 patients with radiographic evidence of hip OA with a K–L score 3 or 4 and T-score ≥ − 2.5 SD. There was no discrepancy for age, sex and comorbidities in the two groups. Specifically, no patient showed oncological, genetic or neurological diseases, whereas more of 80% of patients were affected by hypertension. Body Mass Index (BMI) mean value of OA patients was significantly higher than BMI mean value of OP group (mean value 27.32 ± 4.4 vs 24.56 ± 4.6, p < 0.001) (Table 1). These data confirmed the frequently overweight condition of OA patients .
IL6 expression and localization in skeletal muscle tissue
In order to correlate the inflammatory microenvironment to CLU, we observed IL6 expression in the same tissues by IHC. In muscles tissues from OP patients IL6 was strongly expressed (3 +) in fibers undergoing to atrophy (about 50% of fibers), and a weak signal (±) was present in healthy fibers. Conversely in OA patients, IL6 was uniformly expressed (2 +/3 +) in all tissues examined (Fig. 1a). Since IL6 expression is epigenetically regulated and no data are available on muscle acetylation pattern in OA an OP condition, we observed H4 acetylation state in the same tissues [28, 29].
Histone H4 is acetylated in OP muscle tissues
CLU is strongly expressed in degenerated fibers of OP patients
The presence of CLU was evaluated by IHC in OP and OA muscle tissues. CLU expression was strong in the degenerate fibers of vastus lateralis biopsy samples of OP patients; in fact, CLU was strongly expressed (3 +) in fibers with a diminished diameter as compared to healthy muscle fibers from OP and OA (Fig. 2c). In the latter CLU expression was found diffusely and weakly expressed (1 +) as compared to OP (p < 0.001) (Fig. 2d). It is known that CLU could be also released as a circulating extracellular chaperone. Therefore, in order to verify whether its presence in muscle tissues was due to an endogenous production of the protein or to an intracellular uptake, mRNA was extracted from muscle tissues and from isolated myoblasts from the same patients as previously described. sCLU expression was determined by qRT-PCR and resulted more expressed in OP than OA patients (p < 0.001). This result confirms a potential involvement of the secreted-cytoplasmic isoform of CLU in these two different pathologies (Fig. 2g). In order to study the effect of CLU on proliferation and differentiation in OP and OA, myoblasts were isolated from muscle tissues of the same patients.
CLU is overexpressed in OP myoblasts
Myoblasts obtained were expanded and ICC was performed in order to assess CLU expression at cellular level in the two groups. We observed that CLU is more expressed in OP (Fig. 2e) as compared to OA myoblasts (Fig. 2f). CLU was localized only in the cytoplasm of OP myoblast confirming the results obtained on different patients tissues (Fig. 2c, d). The different expression of sCLU in the two pathologies is shown in Fig. 2h; results obtained are in agreement with data on protein expression previously observed by IHC (Fig. 2a, b), confirming that OP patients express higher level of CLU as compared to OA (p < 0.05).
CLU affects the proliferative rate of OP and OA myoblasts
In order to define the role of CLU in the activation of nuclear factors involved in proliferation, we evaluated the potential activation of NFKB in myoblast conditioned for 6 days with CLU, in order to clarify the long lasting CLU conditioning effect on OP and OA myoblasts (Fig. 3b). In OP untreated myoblast we observed 13% of positive nuclei for NFKB staining and a strong positivity (2 +) in the cytoplasm, suggesting a high amount of the inactive protein. Conversely in OP myoblasts treated with CLU we noticed a decrease of the number of positive nuclei (5.5%) suggesting the lack of activated NFKB protein after treatment (p < 0.05) and concomitantly we observed also a decrease of the cytoplasmic signal (p < 0.01). Differently, in OA untreated myoblasts we observed a diffuse and weak localization of NFKB in the cytoplasm. After 6 days of conditioning an evident increase of NFKB expression in the cytoplasm was observed (p < 0.01).
CLU influences histone acetylation pattern
Epigenetic changes in histone acetylation modulate gene transcription and determine cell fate. Hence, we investigate in OP and OA myoblasts the potential effect of this pleiotropic protein on the acetylation pattern by ICC, with a specific antibody recognizing acetylated histone H4. As shown in Fig. 3, OP untreated myoblasts displayed 90% of positive nuclei for acetyl histone H4 staining (2 +), confirming the results observed in OP tissues (Fig. 2a). In CLU conditioned cultures for 6 days we observed an evident increase of histone H4 acetylation level after 6 days of treatment in 100% of treated cells, suggesting an involvement of CLU in the epigenetic regulation of DNA transcription. Conversely untreated and treated OA myoblasts did not display positivity in the nucleus. The increase of histone H4 acetylation in myoblast and satellite cells has been pointed out as an indicator of differentiation and genes transcription, especially of MyoD related genes . Therefore, in cultured myoblats from OP and OA patients we evaluated the effect of CLU conditioning on a myoblast terminal differentiation marker, MYOG.
CLU modulates MYOG expression
CLU silencing restores the proliferative capability of OP myoblasts
CLU is involved in tissue damage repair in OP disease
CLU silencing affects DNA acetylation and modulates a mediator of the inflammatory response, CX3CR1
Our previous results obtained from exogenous CLU conditioning on OP and OA myoblasts showed a potential involvement of CLU in the epigenetic regulation of DNA, determining a strong increase of histone H4 acetylation in OP myoblasts after 6 days of treatment. We studied OP and OA acetylation pattern also after siRNA transfection for CLU silencing (see “Methods” section). As shown in Fig. 6b, CLU silencing seemed to invert CLU conditioning effect only in OP myoblasts, determining a decrease of positive nuclei for acetyl histone H4 staining, from 53 to 20.2% (p < 0.01), as compared to scramble. No significant modulation occurred in OA transfected cells (Fig. 6b).
OP and OA are two diseases characterized by a strong inflammatory component, especially OA, whose muscle fibers present a strong expression of IL6 (see Fig. 1a), main inflammatory cytokine.
In order to study a possible involvement of CLU in the modulation of the inflammatory response, we analyzed, though ICC, the effect of CLU on CX3CR1 expression. CX3CR1 is a receptor highly expressed on Th1 activated T cells and other activated cell types that, through interaction with its ligand, induces chemotaxis of circulating monocytes and selective recruitment of Th1 lymphocytes responsible of active cronic inflammation [35, 36]. As shown in Fig. 6c, OP myoblasts silenced for CLU gene, showed a decrease of CX3CR1 receptor cytoplasmic expression (p < 0.05). Conversely, CLU silencing determined a strong increase of CX3CR1 expression compared to the scramble (p < 0.01) in OA myoblasts. These results seemed to confirm the potential involvement of CLU in the epigenetic regulation of DNA through histone acetylation and in the inflammatory response.
OP and OA are extremely frequent among elderly people, and their impact on life quality makes them of high social health relevance [37, 38]. The identification of new markers and metabolic targets involved in OP and OA processes is an objective of extreme interest for the discovery of new biological drugs and for the improvement of therapeutic strategies . For the first time, we provided evidences that CLU is strongly expressed in degenerated fibers of OP patients as compared to OA. Moreover, we observed that CLU in OP could be involved in the modulation of histone acetylation and myoblasts terminal differentiation. The latter effect includes inhibition of NFKB nuclear localization and MYOG activation by its nuclear translocation. In fact, the higher level of histone acetylation could be related to the increased expression of genes involved in the activation of satellite cells, to their proliferation and migration towards the atrophic fibers, where they merge and give rise to new regenerated fibers.
Firstly, we characterized the distribution and the expression of the proinflammatory cytokine IL6 in the muscle tissues from the two experimental groups. We observed differences in IL6 expression depending on the severity of the disease and the type of pathology. We found that IL6 was uniformly expressed in OA muscle fibers confirming the strong and diffuse inflammatory state of this pathology. On the contrary, in muscle tissues of OP patients we found a higher expression of IL6 in atrophic fibers and a weaker expression in healthy fibers. According to VanderVeen et al., high circulating levels of IL6 and other proinflammatory citokines, disrupt mitochondrial homeostasis, leading to mitochondria dysfunction and to muscle mass loss during cancer cachexia; in fact, mitochondrial disfunction in skeletal muscle negatively regulates muscle mass. Our previous studies evidenced a link between IL6 and CLU overexpression in highly aggressive tumors, suggesting a possible connection of IL6 derived inflammation with CLU overexpression and neoplastic transformation . We observed that CLU expression was closely related to IL6 presence in muscle tissues. In fact, in OA patients we observed a strong diffused IL6 expression in the sarcoplasm of skeletal muscle tissue, concurring with the chronic inflammatory status of this pathology. In the same patient group we found also a moderate and diffuse presence of CLU, in agreement with data previously published . An overlapping strong expression of IL6 and CLU in fully degenerated or degenerating fibers in muscle tissues of OP patients was noted. Moreover, our in vitro studies on cultures conditioned with CLU have demonstrated a strong involvement of CLU in the downmodulation of cell proliferation and cell death induction after 6 days of treatment, suggesting a potential role of CLU in affecting myoblasts viability. The increased cell proliferation was accompanied also by NFKB downregulation and relocalization in the cytoplasm of OP myoblasts, suggesting a potential network among CLU-NFKB and proliferative arrest. We found a differential histone H4 acetylation pattern in OP and OA tissues. The increased histone H4 acetylation, induced by CLU treatment in OP myoblasts, associated with an active chromatin state, was correlated with a proliferative arrest, as demonstrated by an evident decrease of proliferation and the induction of differentiation exerted by CLU, in OP myoblasts only. The increased histone acetylation during differentiation correlates with the transcription activity of MyoD target genes , and thus suggest that histone acetylation is increased in a subset of genes important for myogenic differentiation.
Accordingly, we observed that CLU affected MYOG expression and localization. In fact the observations on MYOG distribution in untreated OA myoblast indicated that MYOG is located in the cytoplasm only, demonstrating that in OA cells this protein is probably present in an inactive state. CLU is able to induce a light increase of MYOG expression in OA myoblasts only in the cytoplasm. In untreated OP myoblasts, MYOG is present in the cytoplasm as a reservoir and the 80% of nuclei were negative. In OP myoblast cultured in CLU conditioning medium for 6 days, we observed that CLU was able to strongly trigger MYOG translocation from cytoplasm to the nucleus affecting its expression and activation, inducing, as observed phenotipically, terminal differentiation and senescence accompained by cell death. NFKB decrease and inactivation is strongly associated with a decreased proliferation rate in OP, confirming that CLU is operating towards a terminal differentiation and a premature aging state. These results are in agreement with the presence of a strong CLU expression in degenerated fibers as observed in vivo on the tissues of the same patients. Results obtained from CLU silencing experiments confirmed the role of CLU observed also when conditioning OP and OA isolated myoblasts with exogenous CLU. First of all, we noticed that CLU silencing seemed to restore proliferating ability in OP myoblasts only. Conversely, in OA myoblasts, 72 h after transfection, CLU silencing determined a decrease of proliferation. Silencing experiments showed also how the DNA acetylation pattern is reversed in OP myoblasts only. Indeed, OP transfected cells showed a decrease of histone H4 acetylation as compared to OA transfected myoblasts. These data highlights the negative role played by CLU in the osteoporotic disease. Our data evidenced a strong CLU influence on inflammation, through the modulation of CX3CR1, a receptor that, when activated by its ligand causes the chemotaxis of monocytes and the recruitment of Th1 lymphocytes, thus influencing a chronic and active inflammatory state. Hence, CLU silencing in OP strongly reduced the expression of CX3CR1, indicating the potential of CLU to amplify the immune response in this pathology. These data point out a strong correlation between CLU and chronic inflammation typical of the OP-OA condition. In addition, data on CLU silencing in OP demonstrated the effect of CLU also in the activation and expression on TGM2, negatively influencing the tissues damage response. According to the results obtained, CLU, which is strongly expressed in OP patients, seems to exercise a negative effect on the onset and progression of the osteoporotic disease, since its downregulation protects from inflammatory events and restores tissue damage repair ability. Taken together these data strongly suggest that CLU could influence the satellite cells differentiation, inducing a proliferative pulse, cell reset and MYOG activation in OP myoblasts only. Since in muscle tissues of OP patients, as previously published , there is a low reservoir of resident satellite cells, the high level of CLU could induce satellite cells to differentiate, exhausting the satellite cells pool available to repopulate the muscle fibers damaged and leading to a severe sarcopenia. Data obtained in OP myoblasts suggest a possible involvement of CLU in osteoporotic disease, in participating to the loss of satellite cells pool and in massive induction of terminal differentiation and premature senescence. These processes eventually lead to a premature degenerative process and aging, pointing out a potential role of CLU as a new OP diagnostic marker for muscular degeneration and a potential target for specific therapeutic intervention in OP related sarcopenia.
In the present study we observed that CLU, a pleiotropic protein involved in cellular senescence, aging and various age-related diseases, including neurodegeneration, inflammation, vascular damage, diabetes and tumourigenesis, is strongly expressed in the degenerated muscular fibers undergoing atrophy in OP patients. The long lasting somministration of CLU on human isolated myoblasts in vitro induces a proliferative arrest, a modulation of histone acetylation and the nuclear activation of the terminal differentiation marker, MYOG. In addition CLU silencing is able to reduce the expression of CX3CR1, downregulating the local inflammatory response. These data suggest a potential involvement of CLU in the modulation of the inflammation state and in the induction of the premature senescence of osteoporotic myoblasts. Depleting satellite cells pool, causes the state of sarcopenia associated to the osteoporotic disease. Although further studies are warranted to elucidate the exact role of CLU, the present study highlighted the potential action of CLU in osteoporotic disease, suggesting new clinical approaches and strategy for intervention.
SP and UT conceptualized and designed the study. SP carried out the analyses and drafted the initial manuscript. CG, FM, MF, EG, RI and UT participated in sample collection. SP, CG, CP, MCP, MF, MC and UT participated in data interpretation. SP, UT, AO participated in revising manuscript content. All authors agree to be accountable for the work and to ensure that any questions relating to the accuracy and integrity of the paper are investigated and properly resolved. All authors read and approved the final manuscript.
We thank Mario Marini for the technical support. This work was supported by ASI (Italian Space Agency). Project titled “Multidisciplinary Study of the Effects of Microgravity on Bone Cells” call number search DC-DTE-2011-033.
The authors declare that they have no competing interests.
Availability of data and materials
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Ethics approval and consent to participate
The ethics committee of “Policlinico Tor Vergata” (approval reference number # 85/12) approved all experiments described in the present study. All experimental procedures were carried out according to The Code of Ethics of the World Medical Association (Declaration of Helsinki). Informed consent was obtained from all patients prior to surgery. Specimens were handled and carried out in accordance with the approved guidelines.
This work was supported by ASI (Italian Space Agency). Project titled “Multidisciplinary Study of the Effects of Microgravity on Bone Cells” call number search DC-DTE-2011-033.
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