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.

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.

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.

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.

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.

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.

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's 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.

Serology study after BTN162b2 vaccination in participants previously infected with SARS-CoV-2 in two different waves versus naïve.

Background: The antibody response to SARS-CoV-2 mRNA vaccines in individuals with waning immunity generated by a previous SARS-CoV-2 infection, as well as the patterns of IgA and IgM responses in previously infected and in naïve individuals are still poorly understood. Methods: We performed a serology study in a cohort of BTN162b2 mRNA vaccine recipients who were immunologically naïve (N, n = 50) or had been previously infected with SARS-CoV-2 (P.I., n = 51) during the first (n = 25) or second (n = 26) pandemic waves in Italy, respectively. We measured IgG, IgM and IgA antibodies against the SARS-CoV-2 Spike (S) and IgG against the nucleocapsid (N) proteins, as well as the neutralizing activity of sera collected before vaccination, after the first and second dose of vaccine. Results: Most P.I. individuals from the first pandemic wave who showed declining antibody titres responded to the first vaccine dose with IgG-S and pseudovirus neutralization titres that were significantly higher than those observed in N individuals after the second vaccine dose. In all recipients, a single dose of vaccine was sufficient to induce a potent IgA response that was not associated with serum neutralization titres. We observed an unconventional pattern of IgM responses that were elicited in only half of immunologically naïve subjects even after the second vaccine dose. Conclusions: The response to a single dose of vaccine in P.I. individuals is more potent than that observed in N individuals after two doses. Vaccine-induced IgA are not associated with serum neutralization.

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.