Background
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 [
3] 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 [
4]. 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‐
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‐
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‐
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 [
21], 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 [
22]. 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.
Methods
Patients
We enrolled 40 patients who underwent hip surgery in the Orthopedic Department of “Tor Vergata” University Hospital, between 2016 and 2018. Specifically, we enrolled 20 osteoporotic older women who underwent hip arthroplasty for subcapital fractures of the femur (80.58 ± 6.3 years; mean ± standard error of the mean (SEM)) and 20 osteoarthritic older women (67.63 ± 8 years) who underwent hip arthroplasty for OA. Exclusion criteria were history of cancer, myopathies or other neuromuscular diseases or chronic administration of corticosteroid for autoimmune diseases (more than 1 month), diabetes, alcohol abuse, and HBV, HCV, or HIV infections. The main characteristics of OP and OA patients are described in Table
1. All experiments described in the present study were approved by the ethics committee of “Policlinico Tor Vergata” (approval reference number # 85/12). 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.
Table 1
Main characteristics of OP and OA patients
Age | 80.58 ± 6.3 years | 67.63 ± 8 years |
BMI | 24.56 ± 4.6 | 27.32 ± 4.4 |
Menopause age | 42.89 ± 1.3 | 43.68 ± 1.4 |
T-score (L1–L4) | − 2.8 ± 0.7 | − 0.75 ± 0.5 |
T-score (femoral neck) | − 3 ± 0.6 | − 0.67 ± 0.11 |
HHS | – | 47.26 ± 5 |
K–L score | 0–1 | 3/4 |
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 [
23]. 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 [
24]. 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 [
25].
Radiological evaluation
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. [
26] 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.
Sampling
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.
Histology
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.
Immunohistochemistry (IHC)
Serial 5 μm thick sections from formalin-fixed and paraffin-embedded specimens were immunostained for IL6, Clusterin β, Acetyl histone H4 and Myosin-slow (Table
2). After washings, reactions were revealed by HRP-conjugated streoptoavidin method and diaminobenzidine (DAB) incubation. Tissue staining was semi-quantitatively graded for intensity from negative (0) to strong (+++). Slides were independently examined by two pathologists, unaware of the clinical data and molecular results. Moreover, all cases were digitally scanned by iScan (BioImagene, Now Roche-Ventana) with Scanning Resolution 0.46 μm/pixel at ×20. The IHC signals were measured using an automated image analyzing system (MECES). Scoring of immunoreactivity was statistically analyzed by Mann–Whitney’s U test. Twenty sections were stained and analyzed for each experimental group.
Table 2
Primary antibodies used for immunohistochemistry (IHC) analysis
IHC |
Anti-IL6 | Mouse monoclonal, R&D Systems | 1:20 |
Anti-Cluβ | Goat polyclonal, Santa Cruz Biotechnology | 1:100 |
Anti-acetyl histone H4 | Rabbit polyclonal, Upstate | 1:75 |
Anti-myosin slow | Mouse monoclonal, Sigma-Aldrich | 1:100 |
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.
SiRNA transfection
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/cm
2, in 6 well multi-wells at 9000 cell/cm
2 and in 4 well Lab. Tek II Chamber slides at 8000 cell/cm
2, 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
Total RNA was isolated from treated and untreated cells using Tri Reagent (Ambion), according to the manufacturer’s instructions. RNA quantification was performed using spectrophotometry. Reverse transcription of total RNA (1 μg for each myoblast group) was performed with Gene Amp RNA PCR Kit (Applied Biosystems) using Random Examers as primers to cDNA synthesis. β2-microglobulin housekeeping gene was amplified as control. Ethidium-bromide stained 2% agarose gel was run at 100 V and acquired by scanning system. For real time PCR (qRT-PCR) RNA was treated with DNase (2U/ml) and back transcribed with “High Capacity cDNA reverse transcription”. In this study Sybr Green was utilized (Applied Biosystems). GAPDH was used as standard. Each analysis was performed in triplicate. Primers used for cDNA amplification are listed in Table
3.
Table 3
Sequences of primers used for RT-PCR and qRT-PCR
CLU (RT-PCR) | Sense: 5′-GTGCAATGAGACCATGATGG-3′ Antisense: 5′-CAGGTAGTGGTAGGTATCCT-3′ | 55 |
sCLU (qRT-PCR) | Sense: 5′-ATTCTCATCGCTTTGGAAGG-3′ Antisense: 5′-AGACATCAGGGGAGACTTTA-3′ | 58 |
TGM2 (RT-PCR) | Sense: 5′-GAGGAGCTGGTCTTAGAGAGG-3 ́ Antisense: 5′-CGGTCACGACACTGAAGGTG-3 ́ | 62 |
β2-microglobulin (RT-PCR) | Sense: 5′-CTGGAACGGTGAAGGTGACA-3′ Antisense: 5′-AAGGGACTTCCTGTAACAATGCA-3′ | 60 |
GAPDH (qRT-PCR) | Sense: 5′-ACGGATTTGGTCGTATTGG-3′ Antisense: 5′-GATTTTGGAGGGATCTCGC-3′ | 60 |
Immunocytochemistry (ICC)
Human myoblast cells extracted ex vivo were fixed in formalin 10% to perform ICC analysis, as reported above. Primary antibodies used are listed in Table
4. Secondary antibody and following reagents (HRP-conjugated streoptoavidin) were added. After washing, slides were incubated with diaminobenzidine (DAB) and counterstained with haematoxylin.
Table 4
Primary antibodies used for immunocytochemistry (IHC) analysis
ICC |
Anti-Cluβ | Goat polyclonal, Santa Cruz Biotechnology | 1:100 |
Anti-NFKB | Mouse monoclonal, Santa Cruz Biotechnology | 1:100 |
Anti-acetyl histone H4 | Rabbit polyclonal, Upstate | 1:300 |
Anti-CX3CR1 | Rabbit polyclonal, Mo Bi Tec Molecular Biologische Technologie | 1:150 |
Anti-MYOG | Rabbit monoclonal, AbCam | 1:200 |
Statistical analysis
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.
Discussion
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 [
39]. 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 [
14]. 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 [
40]. 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 [
41], 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 [
42], 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.
Conclusions
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.
Authors' contributions
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.
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