Mir-20a regulates in vitro mineralization and BMP signaling pathway by targeting BMP-2 transcript in fish.

MicroRNAs (miRNAs) are important regulators of vertebrate development but their role during skeletogenesis remains unknown. In this regard, we investigated the mineralogenic activity of miR-20a, a miRNA associated with osteogenesis, in fish bone-derived cells. Expression of miR-20a was up-regulated during differentiation and its overexpression inhibited mineralization, suggesting a role in fish tissue calcification. In this regard, a conserved miR-20a binding site was identified in bone morphogenetic protein 2 (BMP-2) 3'UTR and its functionality was evidenced through luciferase assays, and further confirmed by western-blot and qPCR. Type II BMP receptor (BMPR2) is also targeted by miR-20a in mammalian systems and evidence was collected for the presence of a binding site in fish sequences. We propose that miR-20a is a regulator of BMP pathway through specific action on BMP-2 and possibly BMPR2. Overexpression of miR-20a was also shown to up-regulate matrix Gla protein (MGP) transcript, a physiological inhibitor of calcification previously found to form a complex with BMP-2. We propose that MGP may play a role in the anti-mineralogenic effect promoted by miR-20a by decreasing availability of BMP-2. This study gives new insights into miRNA-mediated regulation of BMP-2, and sheds light into the potential role of miR-20a as a regulator of skeletogenesis.

Antioxidant, Mineralogenic and Osteogenic Activities of Spartina alterniflora and Salicornia fragilis Extracts Rich in Polyphenols.

Osteoporosis is an aging-related disease and a worldwide health issue. Current therapeutics have failed to reduce the prevalence of osteoporosis in the human population, thus the discovery of compounds with bone anabolic properties that could be the basis of next generation drugs is a priority. Marine plants contain a wide range of bioactive compounds and the presence of osteoactive phytochemicals was investigated in two halophytes collected in Brittany (France): the invasive Spartina alterniflora and the native Salicornia fragilis. Two semi-purified fractions, prepared through liquid-liquid extraction, were assessed for phenolic and flavonoid contents, and for the presence of antioxidant, mineralogenic and osteogenic bioactivities. Ethyl acetate fraction (EAF) was rich in phenolic compounds and exhibited the highest antioxidant activity. While S. fragilis EAF only triggered a weak proliferative effect in vitro, S. alterniflora EAF potently induced extracellular matrix mineralization (7-fold at 250 µg/mL). A strong osteogenic effect was also observed in vivo using zebrafish operculum assay (2.5-fold at 10 µg/mL in 9-dpf larvae). Results indicate that polyphenol rich EAF of S. alterniflora has both antioxidant and bone anabolic activities. As an invasive species, this marine plant may represent a sustainable source of molecules for therapeutic applications in bone disorders.

Circulating small non-coding RNAs provide new insights into vitamin K nutrition and reproductive physiology in teleost fish.

BACKGROUND: Vitamin K (VK) is a fat-soluble vitamin known for its essential role in blood coagulation, but also on other biological processes (e.g. reproduction, brain and bone development) have been recently suggested. Nevertheless, the molecular mechanisms behind its particular function on reproduction are not yet fully understood. METHODS: The potential role of VK on reproduction through nutritional supplementation in Senegalese sole (Solea senegalensis) was assessed by gonadal maturation and 11-ketosterone, testosterone and estriol plasma levels when fed with control or VK supplemented (1250 mg kg-1 of VK1) diets along a six month trial. At the end, sperm production and quality (viability and DNA fragmentation) were evaluated. Circulating small non-coding RNAs (sncRNAs) in blood plasma from males were also studied through RNA-Seq. RESULTS: Fish fed with dietary VK supplementation had increased testosterone levels and lower sperm DNA fragmentation. SncRNAs from blood plasma were found differentially expressed when nutritional and sperm quality conditions were compared. PiR-675//676//4794//5462 and piR-74614 were found up-regulated in males fed with dietary VK supplementation. Let-7g, let-7e(18nt), let-7a-1, let-7a-3//7a-2//7a-1, let-7e(23nt) and piR-675//676//4794//5462 were found to be up-regulated and miR-146a and miR-146a-1//146a-2//146a-3 down-regulated when fish with low and high sperm DNA fragmentation were compared. Bioinformatic analyses of predicted mRNAs targeted by sncRNAs revealed the potential underlying pathways. CONCLUSIONS: VK supplementation improves fish gonad maturation and sperm quality, suggesting an unexpected and complex regulation of the nutritional status and reproductive performance through circulating sncRNAs. GENERAL SIGNIFICANCE: The use of circulating sncRNAs as reliable and less-invasive physiological biomarkers in fish nutrition and reproduction has been unveiled.

The xenobiotic sensor PXR in a marine flatfish species (Solea senegalensis): Gene expression patterns and its regulation under different physiological conditions.

The pregnane X receptor (PXR) is a nuclear receptor belonging to the NR1I sub-family and a known master regulator of xenobiotic metabolism. New roles have been recently proposed in mammals through its activation by vitamin K (VK) such as regulation of glucose metabolism, bone homeostasis, reproduction, neuronal development and cognitive capacities. In marine fish species little is known about PXR and its potential roles. Here, expression patterns of pxr transcripts and conservation of protein domains were determined in the Senegalese sole (Solea senegalensis), a marine flatfish model species in aquatic ecotoxicology. In addition to a full coding sequence transcript (sspxr1), two variants lacking DNA and/or ligand binding domains (sspxr2 and sspxr3) were also identified. The expression of sspxr1 during early development and in adult tissues was ubiquitous, but highest levels were observed in liver, intestine and skin. Expression was also detected by in situ hybridization in chondrocytes and cells from the granular and inner nuclear layers in three month old fish. Finally, sspxr1 expression was shown to be differentially regulated under physiological conditions related with fasting, VK and warfarin metabolism. The present work provides new and basic knowledge regarding pxr sequence and expression patterns in a marine flatfish species to unveil the potential impact of xenobiotics on marine fish physiology, and will allow a better and more ecosystemic environmental risk assessment of different pollutants over the marine environments with the development of reporter assays using PXR sequences from evolutionary distantly marine species (such as vertebrate and invertebrate marine species).