Modulation of miR-204 expression during chondrogenesis

RUNX2 and SOX9 are two pivotal transcriptional regulators of chondrogenesis. It has been demonstrated that RUNX2 and SOX9 physically interact; RUNX2 transactivation may be inhibited by SOX9. In addition, RUNX2 exerts reciprocal inhibition on SOX9 transactivity. Epigenetic control of gene expression plays a major role in the alternative differentiation fates of stem cells; in particular, it has been reported that SOX9 can promote the expression of miRNA (miR)-204. Our aim was therefore to investigate the miR-204-5p role during chondrogenesis and to identify the relationship between this miR and the transcription factors plus downstream genes involved in chondrogenic commitment and differentiation. To evaluate the role of miR-204 in chondrogenesis, we performed in vitro transfection experiments by using Mesenchymal Stem Cells (MSCs). We also evaluated miR-204-5p expression in zebrafish models (adults and larvae). By silencing miR-204 during the early differentiation phase, we observed the upregulation of SOX9 and chondrogenic related genes compared to controls. In addition, we observed the upregulation of COL1A1 (a RUNX2 downstream gene), whereas RUNX2 expression of RUNX2 was slightly affected compared to controls. However, RUNX2 protein levels increased in miR-204-silenced cells. The positive effects of miR204 silencing on osteogenic differentiation were also observed in the intermediate phase of osteogenic differentiation. On the contrary, chondrocytes' maturation was considerably affected by miR-204 downregulation. In conclusion, our results suggest that miR-204 negatively regulates the osteochondrogenic commitment of MSCs, while it positively regulates chondrocytes' maturation

Two novel C-terminus RUNX2 mutations in two cleidocranial dysplasia (CCD) patients impairing p53 expression

Cleidocranial dysplasia (CCD), a dominantly inherited skeletal disease, is characterized by a variable phenotype ranging from dental alterations to severe skeletal defects. Either de novo or inherited mutations in the RUNX2 gene have been identified in most CCD patients. Transcription factor RUNX2, the osteogenic master gene, plays a central role in the commitment of mesenchymal stem cells to osteoblast lineage. With the aim to analyse the effects of RUNX2 mutations in CCD patients, we investigated RUNX2 gene expression and the osteogenic potential of two CCD patients’ cells. In addition, with the aim to better understand how RUNX2 mutations interfere with osteogenic differentiation, we performed string analyses to identify proteins interacting with RUNX2 and analysed p53 expression levels. Our findings demonstrated for the first time that, in addition to the alteration of downstream gene expression, RUNX2 mutations impair p53 expression affecting osteogenic maturation. In conclusion, the present work provides new insights into the role of RUNX2 mutations in CCD patients and suggests that an in-depth analysis of the RUNX2-associated gene network may contribute to better understand the complex molecular and phenotypic alterations in mutant subjects

Effects of a 4400 km ultra-cycling non-competitive race and related training on body composition and circulating progenitors differentiation

Background: NorthCape4000 (NC4000) is the most participated ultra-endurance cycling race. Eight healthy male Caucasian amateur cyclists were evaluated: (a) before starting the preparation period; (b) in the week preceding NC4000 (after the training period); (c) after NC4000 race, with the aim to identify the effects of ultra-cycling on body composition, aerobic capacity and biochemical parameters as well as on the differentiation of progenitor cells. Methods: Bioelectrical impedance analysis (BIA) and dual energy x-ray absorptiometry (DEXA) assessed body composition; cardiopulmonary exercise test (CPET) evaluated aerobic capacity. Differentiation of circulating progenitor cells was evaluated by analyzing the modulation in the expression of relevant transcription factors. In addition, in vitro experiments were performed to investigate the effects of sera of NC4000 participants on adipogenesis and myogenesis. The effects of NC4000 sera on Sestrins and Sirtuin modulation and the promotion of brown adipogenesis in progenitor cells was investigated as well. Two-tailed Student's paired-test was used to perform statistical analyses. Results: We observed fat mass decrease after training as well as after NC4000 performance; we also recorded that vitamin D and lipid profiles were affected by ultra-cycling. In addition, our findings demonstrated that post-NC4000 participant's pooled sera exerted a positive effect in stimulating myogenesis and in inducing brown adipogenesis in progenitor cells. Conclusions: The training program and Ultra-cycling lead to beneficial effects on body composition and biochemical lipid parameters, as well as changes in differentiation of progenitor cells, with significant increases in brown adipogenesis and in MYOD levels

Physical activity modulates miRNAs levels and enhances MYOD expression in myoblasts

Stem cells functions are regulated by different factors and non-conding RNAs, such as microRNA. MiRNAsplay an important role in modulating the expression of genes involved in the commitment and differentiation of progenitor cells. MiRNAs are post transcriptional regulators which may be modulated by physical exercise. MiRNAs, by regulating different signaling pathways, play an important role in myogenesis as well as in muscle activity. MiRNAs quantification may be considered for evaluating physical performance or muscle recovery. With the aim to identify specific miRNAs potentially involved in myogenesis and modulated by physical activity, we investigated miRNAs expression following physical performance in Peripheral Blood Mononuclear Cells (PBMCs) and in sera of half marathon (HM) runnners. The effect of runners sera on Myogenesis in in vitro cellular models was also explored. Therefore, we performed Microarray Analysis and Real Time PCR assays, as well as in vitro cell cultures analysis to investigate myogenic differentiation. Our data demonstrated gender-specific expression patterns of PBMC miRNAs before physical performance. In particular, miR223-3p, miR26b-5p, miR150-5p and miR15-5p expression was higher, while miR7a-5p and miR7i-5p expression was lower in females compared to males. After HM, miR152-3p, miR143-3p, miR27a-3p levels increased while miR30b-3p decreased in both females and males: circulating miRNAs mirrored these modulations. Furthermore, we also observed that the addition of post-HM participants sera to cell cultures exerted a positive effect in stimulating myogenesis. In conclusion, our data suggest that physical activity induces the modulation of myogenesis-associated miRNAs in bothfemales and males, despite the gender-associated different expression of certain miRNAs, Noteworthy, these findings might be useful for evaluating potential targets for microRNA based-therapies in diseases affecting the myogenic stem cells population

Fisetin: an integrated approach to identify a strategy promoting osteogenesis

Flavonoids may modulate the bone formation process. Among flavonoids, fisetin is known to counteract tumor growth, osteoarthritis, and rheumatoid arthritis. In addition, fisetin prevents inflammation-induced bone loss. In order to evaluate its favorable use in osteogenesis, we assayed fisetin supplementation in both in vitro and in vivo models and gathered information on nanoparticle-mediated delivery of fisetin in vitro and in a microfluidic system. Real-time RT-PCR, Western blotting, and nanoparticle synthesis were performed to evaluate the effects of fisetin in vitro, in the zebrafish model, and in ex vivo samples. Our results demonstrated that fisetin at 2.5 μM concentration promotes bone formation in vitro and mineralization in the zebrafish model. In addition, we found that fisetin stimulates osteoblast maturation in cell cultures obtained from cleidocranial dysplasia patients. Remarkably, PLGA nanoparticles increased fisetin stability and, consequently, its stimulating effects on RUNX2 and its downstream gene SP7 expression. Therefore, our findings demonstrated the positive effects of fisetin on osteogenesis and suggest that patients affected by skeletal diseases, both of genetic and metabolic origins, may actually benefit from fisetin supplementation

Expression of FBXW11 in normal and disease-associated osteogenic cells

The ubiquitin-proteasome system (UPS) plays an important role in maintaining cellular homeostasis by degrading a multitude of key regulatory proteins. FBXW11, also known as b-TrCP2, belongs to the F-box family, which targets the proteins to be degraded by UPS. Transcription factors or proteins associated with cell cycle can be modulated by FBXW11, which may stimulate or inhibit cellular proliferation. Although FBXW11 has been investigated in embryogenesis and cancer, its expression has not been evaluated in osteogenic cells. With the aim to explore FBXW11gene expression modulation in the osteogenic lineage we performed molecular investigations in mesenchymal stem cells (MSCs) and osteogenic cells in normal and pathological conditions. In vitro experiments as well as ex vivo investigations have been performed. In particular, we explored the FBXW11 expression in normal osteogenic cells as well as in cells of cleidocranial dysplasia (CCD) patients or osteosarcoma cells. Our data showed that FBXW11 expression is modulated during osteogenesis and overexpressed in circulating MSCs and in osteogenically stimulated cells of CCD patients. In addition, FBXW11 is post-transcriptionally regulated in osteosarcoma cells leading to increased levels of beta-catenin. In conclusion, our findings show the modulation of FBXW11 in osteogenic lineage and its dysregulation in impaired osteogenic cells

A potential role of RUNX2- RUNT domain in modulating the expression of genes involved in bone metastases: an in vitro study with melanoma cells

Ectopic expression of RUNX2 has been reported in several tumors. In melanoma cells, the RUNT domain of RUNX2 increases cell proliferation and migration. Due to the strong link between RUNX2 and skeletal development, we hypothesized that the RUNT domain may be involved in the modulation of mechanisms associated with melanoma bone metastasis. Therefore, we evaluated the expression of metastatic targets in wild type (WT) and RUNT KO melanoma cells by array and real-time PCR analyses. Western blot, ELISA, immunofluorescence, migration and invasion ability assays were also performed. Our findings showed that the expression levels of bone sialoprotein (BSP) and osteopontin (SPP1) genes, which are involved in malignancy-induced hypercalcemia, were reduced in RUNT KO cells. In addition, released PTHrP levels were lower in RUNT KO cells than in WT cells. The RUNT domain also contributes to increased osteotropism and bone invasion in melanoma cells. Importantly, we found that the ERK/p-ERK and AKT/p-AKT pathways are involved in RUNT-promoted bone metastases. On the basis of our findings, we concluded that the RUNX2 RUNT domain is involved in the mechanisms promoting bone metastasis of melanoma cells via complex interactions between multiple players involved in bone remodeling

Modulation of miR-146b Expression during Aging and the Impact of Physical Activity on Its Expression and Chondrogenic Progenitors

The finding of molecules associated with aging is important for the prevention of chronic degenerative diseases and for longevity strategies. MicroRNAs (miRNAs) are post-transcriptional regulators involved in many biological processes and miR-146b-5p has been shown to be involved in different degenerative diseases. However, miR-146b-5p modulation has not been evaluated in mesenchymal stem cells (MSCs) commitment or during aging. Therefore, the modulation of miR-146b-5p in the commitment and differentiation of mesenchymal cells as well as during maturation and aging in zebrafish model were analyzed. In addition, circulating miR-146b-5p was evaluated in human subjects at different age ranges. Thus, the role of physical activity in the modulation of miR-146b-5p was also investigated. To achieve these aims, RT (real-time)-PCR, Western blot, cell transfections, and three-dimensional (3D) culture techniques were applied. Our findings show that miR-146b-5p expression drives MSCs to adipogenic differentiation and increases during zebrafish maturation and aging. In addition, miR-146b-5p expression is higher in females compared to males and it is associated with the aging in humans. Interestingly, we also observed that the physical activity of walking downregulates circulating miR-146b-5p levels in human females and increases the number of chondroprogenitors. In conclusion, miR-146b-5p can be considered an age-related marker and can represent a useful marker for identifying strategies, such as physical activity, aimed at counteracting the degenerative processes of aging

Role of miR-204 and bioactive compounds in modulating chondrogenic and osteogenic differentiation of mesenchymal stem cells

Nelle malattie associate all'invecchiamento, come l'osteoporosi e l'artrosi, la cartilagine e l'osso sono i tessuti più comunemente danneggiati. I condrociti e gli osteociti originano da cellule staminali mesenchimali pluripotenti (MSCs) attraverso il differenziamento condrogenico e osteogenico; due processi correlati. RUNX2 e SOX9 sono i principali fattori di trascrizione per l’osteogenesi e la condrogenesi, rispettivamente. Essi interagiscono direttamente e possono inibire reciprocamente la loro attivazione. La regolazione epigenetica mediata dai miRNA influenza notevolmente la direzione del differenziamento delle MSCs. È stato scoperto che il miR-204 ha come bersaglio molecolare l’mRNA di RUNX2 e che la sua espressione è promossa da SOX9. È risaputo che lo stato di infiammazione altera il differenziamento condrogenico e osteogenico delle MSCs. Il metilsulfonilmetano (MSM) e la fisetina, due composti nutraceutici con proprietà anti-infiammatorie e antiossidanti, sono efficaci nella gestione terapeutica delle malattie degenerative della cartilagine e dell'osso. In questo studio, abbiamo delucidato il ruolo del miR-204 durante il commissionamento e il differenziamento delle MSCs e analizzato gli effetti complessivi dell’MSM e della fisetina sulla condrogenesi e osteogenesi. Le analisi sperimentali sono state condotte sia in vitro che in vivo. Il silenziamento dell’attività del miR-204 nelle MSCs aumentava l’espressione di geni associati alla maturazione condrogenica (SOX9, COMP, COL2A1) e osteogenica (COL1A1) durante la fase iniziale del differenziamento delle MSCs. Al contrario, l’assenza di attività del miR-204 influenzava notevolmente la maturazione dei condroblasti e degli osteoblasti durante la fase tardiva del differenziamento. L’espressione di RUNX2 risultava leggermente alterata rispetto al controllo, mentre il livello di espressione della proteina aumentava nelle cellule in cui il miR-204 era silenziato. In vivo, l’espressione del miR-204 aumentava durante lo sviluppo larvale degli zebrafish e fino a 6 mesi di età, per poi diminuire quando gli zebrafish anziani. È interessante notare come i livelli di espressione della proteina -catenina e di p-ERK diminuivano durante il differenziamento condrogenico delle MSCs, mentre invece negli zebrafish aventi 2 anni di età alti livelli di p-ERK e bassi livelli di aggrecano, un marcatore di maturazione condrogenica, suggerivano un ridotto processo di condrogenesi. In seguito a trattamento con IL-1, l’espressione del miR-204 e di SOX9 diminuiva sia nelle MSCs che nei condrociti maturi. È interessante notare come l’aggiunta dell’MSM abbia annullato gli effetti negativi dell’IL-1 in entrambi i tipi cellulari aumentando l’espressione di miR-204, SOX9, SESN1 e COMP. L’MSM promuoveva la condrogenesi sia nelle larve che negli zebrafish adulti. Inoltre, l’MSM stimolava la maturazione degli osteoblasti in vitro e l’osteogenesi in vivo, come dimostrato dalla maggiore intensità della colorazione con calceina e Alizarin Red (AR) negli zebrafish adulti e nelle larve trattate con MSM rispetto ai non trattati. Inoltre i livelli di p-ERK erano più bassi nelle pinne e nelle squame degli zebrafish trattati con MSM rispetto ai non trattati. Il trattamento con fisetina promuoveva il differenziamento osteogenico delle MSCs. Inoltre, la fisetina favoriva la formazione e la mineralizzazione dell’osso in vivo, come evidenziato dalla maggiore intensità della colorazione con calceina e AR negli zebrafish adulti e nelle larve trattate con fisetina rispetto ai non trattati. Sorprendentemente, la fisetina aumentava sensibilmente la maturazione osteogenica di fibroblasti primari isolati da pazienti affetti da Displasia Cleidocranica, una malattia genetica dello sviluppo osseo causata da mutazioni nel gene RUNX2. Abbiamo generato delle nanoparticelle costituite da acido lattico e glicolico (PLGA) con incapsulata la fisetina PLGA-(Fis). Sorprendentemente, le nanoparticelle PLGA hanno migliorato la stabilità della fisetina, amplificando i suoi effetti sull’espressione genica. Inoltre, mediante un esperimento di microfluidica organ-on-a-chip, abbiamo dimostrato come PLGA-(Fis) erano in grado di attraversare l’epitelio intestinale umano preservando la fisetina dalla degradazione. In conclusione, questo studio evidenzia come il miR-204 regola negativamente il commissionamento osteo-condrogenico delle MSCs, mentre regola positivamente la maturazione dei condroblasti e degli osteoblasti. Inoltre, i dati sperimentali dimostrano come l’MSM e la fisetina agiscano influenzando il commissionamento e il differenziamento delle MSCs e hanno effetti positivi sull’osteogenesi, suggerendo che pazienti affetti da malattie scheletriche sia di origine genetica che metabolica possono trarre beneficio dalla loro somministrazione.In aging-related diseases, such as osteoporosis and osteoarthritis (OA), cartilage and bone are the most commonly damaged tissues. Chondrocytes and osteocytes originate from pluripotent mesenchymal stem cells (MSCs) through chondrogenic and osteogenic differentiation; two interrelated processes. RUNX2 and SOX9 are the major transcription factors for osteogenesis and chondrogenesis, respectively. They directly interact and can inhibit each other’s transactivation. Epigenetic regulation by miRNA greatly influences the differentiation fate of MSCs. It has been found that miR-204 binds to the 3’-UTR of RUNX2 mRNA and that its expression is promoted by SOX9. Inflammation has been shown to impair MSCs chondrogenic and osteogenic differentiation. Methylsulfonylmethane (MSM) and fisetin, two nutraceutical compounds with anti-inflammatory and antioxidant properties, have been proved to be effective in the management of cartilage and bone degenerative disorders. In this study, we elucidated the miR-204 role during MSCs’ commitment and differentiation steps and investigated the overall effects of MSM and fisetin on chondrogenesis and osteogenesis. The analyses were conducted both in vitro and in vivo. The silencing of miR-204 activity in MSCs enhanced the expression of chondrogenic (SOX9, COMP, COL2A1) and osteogenic (COL1A1) maturation-related genes during the early MSCs differentiation phase. On the contrary, the absence of miR-204 activity considerably affected the chondroblasts’ and osteoblasts’ maturation during the late MSCs differentiation phase. RUNX2 expression was slightly altered in miR-204 silenced cells compared to control, although RUNX2 protein levels were increased. In vivo, miR-204 expression increased during zebrafish larval development and up to six months of growth, but then it dropped as zebrafish grew older. Interestingly, β-catenin and p-ERK protein levels decreased during in vitro MSCs chondrogenic differentiation. Conversely, in 2 years old zebrafish, high levels of p-ERK and low levels of aggrecan, a chondrogenic maturation marker, suggested a reduced chondrogenic commitment. Upon IL-1β treatment, miR-204 and SOX9 expression decreased in MSCs and mature chondrocytes. Interestingly, the addition of MSM reversed IL-1β effects in both cell types by increasing miR-204, SOX9, SESN1 and COMP expression. MSM promoted chondrogenesis in both adult zebrafish and larvae. Moreover, MSM stimulated osteoblasts maturation in vitro and osteogenesis in vivo, as shown by the more intense calcein and Alizarin Red (AR) staining in MSM-treated adult zebrafish and larvae compared to untreated. Interestingly, p-ERK levels were lower in the fins and scales of MSM-treated zebrafish compared to untreated. Fisetin supplementation promoted MSCs osteogenic differentiation in vitro. Moreover, fisetin stimulated bone formation and mineralization in vivo, as shown by the higher AR and calcein staining in fisetin-treated adult zebrafish and larvae compared to untreated. Remarkably, fisetin significantly enhanced osteogenic maturation in primary fibroblasts of patients with Cleidocranial Dysplasia, a genetic skeletal disease caused by RUNX2 mutations. A delivery system with PLGA (poly lactic-co-glycolic acid) nanoparticles was generated to encapsulate fisetin [PLGA-(Fis)]. Surprisingly, PLGA nanoparticles improved fisetin stability and its biological effects on gene expression. Moreover, in a microfluidic organ-on-a-chip experiment, PLGA-(Fis) nanoparticles showed the ability to cross the human intestinal epithelial tissue, preventing fisetin degradation. In conclusion, this study shows that miR-204 negatively regulates the MSCs’ commitment to the osteochondrogenic lineage, while it positively influences the maturation of chondroblasts and osteoblasts. Moreover, our data show that MSM and fisetin act by influencing MSC commitment and differentiation and have beneficial effects on osteogenesis, suggesting that patients affected by skeletal degenerative diseases of both genetic and metabolic origins may benefit from their administration

Methylsulfonylmethane enhances MSC chondrogenic commitment and promotes pre-osteoblasts formation

Background: Methylsulfonylmethane (MSM) is a nutraceutical compound which has been indicated to counteract osteoarthritis, a cartilage degenerative disorder. In addition, MSM has also been shown to increase osteoblast differentiation. So far, few studies have investigated MSM role in the differentiation of mesenchymal stem cells (MSCs), and no study has been performed to evaluate its overall effects on both osteogenic and chondrogenic differentiation. These two mutually regulated processes share the same progenitor cells. Methods: Therefore, with the aim to evaluate the effects of MSM on chondrogenesis and osteogenesis, we analyzed the expression of SOX9, RUNX2, and SP7 transcription factors in vitro (mesenchymal stem cells and chondrocytes cell lines) and in vivo (zebrafish model). Real-time PCR as well Western blotting, immunofluorescence, and specific in vitro and in vivo staining have been performed. Student\u2019s paired t test was used to compare the variation between the groups. Results: Our data demonstrated that MSM modulates the expression of differentiation-related genes both in vitro and in vivo. The increased SOX9 expression suggests that MSM promotes chondrogenesis in treated samples. In addition, RUNX2 expression was not particularly affected by MSM while SP7 expression increased in all MSM samples/model analyzed. As SP7 is required for the final commitment of progenitors to preosteoblasts, our data suggest a role of MSM in promoting preosteoblast formation. In addition, we observed a reduced expression of the osteoclast-surface receptor RANK in larvae and in scales as well as a reduced pERK/ERK ratio in fin and scale of MSM treated zebrafish. Conclusions: In conclusion, our study provides new insights into MSM mode of action and suggests that MSM is a useful tool to counteract skeletal degenerative diseases by targeting MSC commitment and differentiation. Keywords: Methylsulfonylmethane, Mesenchymal stem cells, Chondrocytes, Osteoblast