Skeletal diseases caused by mutations in PTH1R show aberrant differentiation of skeletal progenitors due to dysregulation of DEPTOR

Alterations in the balance between skeletogenesis and adipogenesis is a pathogenic feature in multiple skeletal disorders. Clinically, enhanced bone marrow adiposity in bones impairs mobility and increases fracture risk, reducing the quality of life of patients. The molecular mechanism that underlies the balance between skeletogenesis and adipogenesis is not completely understood but alterations in skeletal progenitor cells’ differentiation pathway plays a key role. We recently demonstrated that parathyroid hormone (PTH)/PTH-related peptide (PTHrP) control the levels of DEPTOR, an inhibitor of the mechanistic target of rapamycin (mTOR), and that DEPTOR levels are altered in different skeletal diseases. Here, we show that mutations in the PTH receptor-1 (PTH1R) alter the differentiation of skeletal progenitors in two different skeletal genetic disorders and lead to accumulation of fat or cartilage in bones. Mechanistically, DEPTOR controls the subcellular localization of TAZ (transcriptional co-activator with a PDZ-binding domain), a transcriptional regulator that governs skeletal stem cells differentiation into either bone and fat. We show that DEPTOR regulation of TAZ localization is achieved through the control of Dishevelled2 (DVL2) phosphorylation and that it ultimately translates into the transcriptional control of PTH and WNT target genes. Depending on nutrient availability, DEPTOR directly interacts with PTH1R to regulate PTH/PTHrP signaling or it forms a complex with TAZ, to prevent its translocation to the nucleus and therefore inhibit its transcriptional activity. Our data point DEPTOR as a key molecule in skeletal progenitor differentiation; its dysregulation under pathologic conditions results in aberrant bone/fat balance.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Bone organoid generation based on double scaffolding.

Decellularized bone matrix ( is a classic approach in bone tissue engineering based on the removal of bone tissue cells and the calcified phase using chemical, physical o enzymatic agents The resulting matrix preserves its three dimensional structure and biomechanical properties, providing a native microenvironment suitable for osteogenic development However, due to the complexity of the bone structure, with this method has not been possible to obtain an experimentally reproducible bone organoid that sufficiently replicates the bone biology Another approach to generate organoids is the use of hydrogels functionalized with adhesion peptides and morphogenetic proteins that favor the differentiation and osteogenic capacity of the organoid In this work we developed a new bone model based on the combination of hydrogels and DBMs We tested PVA and dextran hydrogels in combination with BMP responsive reporter cell line ( and the long term effect of the RGD peptide addition to hydrogels evaluating osteogenic differentiation by alkaline phosphatase method and luciferase assays We analyze by histology decellularized DBMs combined with dextran hydrogels containing BRITER cells cultured under osteogenic conditions for 3 weeks Our results showed that this double scaffolding is able to support osteogenic differentiation as well as osseointegration, proving its potential for bone organoid generationUniversidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The Skeletal Atlas: A holistic approach to transcriptomics in the skeleton.

Gene expression atlases are powerful bioinformatics tools that allow access to the scientific community to explore omics data. In biomedicine, numerous gene expression databases are available, yet none provide enough information about localized transcriptomics in the skeletal system. In this work we try to solve this gap of knowledge by creating a transcriptomic atlas which brings together different works across the skeletal system, analyzing several key tissue compartments in normal and pathological conditions from mouse and human after analyzing more than 200 studies. Our atlas shows a user-friendly representation which allows the comparison of multiple genes across the different tissues of the skeleton: bone, cartilage, tendon, ligament, growth plate and skeletal muscle. As expected, our first finding was the identification of a significant heterogeneity between experiments from similar studies but from different labs, so we have created a dataset processing pipeline with inclusive/exclusive criteria of RNAseq experiments based in sequencing quality and purity of the analyzed tissues in search of a normalization protocol. We believe that this atlas will allow the homogenization of the public transcriptomic studies in the skeleton and will settle the basis to incorporate the upcoming data from single cell transcriptomics studies.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

Skeletal diseases caused by mutations in PTH1R show aberrant differentiation of skeletal progenitors due to dysregulation of DEPTOR

Alterations in the balance between skeletogenesis and adipogenesis is a pathogenic feature in multiple skeletal disorders. Clinically, enhanced bone marrow adiposity in bones impairs mobility and increases fracture risk, reducing the quality of life of patients. The molecular mechanism that underlies the balance between skeletogenesis and adipogenesis is not completely understood but alterations in skeletal progenitor cells’ differentiation pathway plays a key role. We recently demonstrated that parathyroid hormone (PTH)/PTH-related peptide (PTHrP) control the levels of DEPTOR, an inhibitor of the mechanistic target of rapamycin (mTOR), and that DEPTOR levels are altered in different skeletal diseases. Here, we show that mutations in the PTH receptor-1 (PTH1R) alter the differentiation of skeletal progenitors in two different skeletal genetic disorders and lead to accumulation of fat or cartilage in bones. Mechanistically, DEPTOR controls the subcellular localization of TAZ (transcriptional co-activator with a PDZ-binding domain), a transcriptional regulator that governs skeletal stem cells differentiation into either bone and fat. We show that DEPTOR regulation of TAZ localization is achieved through the control of Dishevelled2 (DVL2) phosphorylation. Depending on nutrient availability, DEPTOR directly interacts with PTH1R to regulate PTH/PTHrP signaling or it forms a complex with TAZ, to prevent its translocation to the nucleus and therefore inhibit its transcriptional activity. Our data point DEPTOR as a key molecule in skeletal progenitor differentiation; its dysregulation under pathologic conditions results in aberrant bone/fat balance

Generation and analysis of ESTs from strawberry (Fragaria xananassa) fruits and evaluation of their utility in genetic and molecular studies

<p>Abstract</p> <p>Background</p> <p>Cultivated strawberry is a hybrid octoploid species (<it>Fragaria xananassa </it>Duchesne ex. Rozier) whose fruit is highly appreciated due to its organoleptic properties and health benefits. Despite recent studies on the control of its growth and ripening processes, information about the role played by different hormones on these processes remains elusive. Further advancement of this knowledge is hampered by the limited sequence information on genes from this species, despite the abundant information available on genes from the wild diploid relative <it>Fragaria vesca</it>. However, the diploid species, or one ancestor, only partially contributes to the genome of the cultivated octoploid. We have produced a collection of expressed sequence tags (ESTs) from different cDNA libraries prepared from different fruit parts and developmental stages. The collection has been analysed and the sequence information used to explore the involvement of different hormones in fruit developmental processes, and for the comparison of transcripts in the receptacle of ripe fruits of diploid and octoploid species. The study is particularly important since the commercial fruit is indeed an enlarged flower receptacle with the true fruits, the achenes, on the surface and connected through a network of vascular vessels to the central pith.</p> <p>Results</p> <p>We have sequenced over 4,500 ESTs from <it>Fragaria xananassa</it>, thus doubling the number of ESTs available in the GenBank of this species. We then assembled this information together with that available from <it>F. xananassa </it>resulting a total of 7,096 unigenes. The identification of SSRs and SNPs in many of the ESTs allowed their conversion into functional molecular markers. The availability of libraries prepared from green growing fruits has allowed the cloning of cDNAs encoding for genes of auxin, ethylene and brassinosteroid signalling processes, followed by expression studies in selected fruit parts and developmental stages. In addition, the sequence information generated in the project, jointly with previous information on sequences from both <it>F. xananassa </it>and <it>F. vesca</it>, has allowed designing an oligo-based microarray that has been used to compare the transcriptome of the ripe receptacle of the diploid and octoploid species. Comparison of the transcriptomes, grouping the genes by biological processes, points to differences being quantitative rather than qualitative.</p> <p>Conclusions</p> <p>The present study generates essential knowledge and molecular tools that will be useful in improving investigations at the molecular level in cultivated strawberry (<it>F. xananassa</it>). This knowledge is likely to provide useful resources in the ongoing breeding programs. The sequence information has already allowed the development of molecular markers that have been applied to germplasm characterization and could be eventually used in QTL analysis. Massive transcription analysis can be of utility to target specific genes to be further studied, by their involvement in the different plant developmental processes.</p

DEPTOR in Skeletal Development and Diseases

Discovered in 2009, the DEP-domain containing mTOR-interacting protein, DEPTOR, is a known regulator of the mechanistic target of rapamycin (mTOR), an evolutionarily conserved kinase that regulates diverse cellular processes in response to environmental stimuli. DEPTOR was originally identified as a negative regulator of mTOR complexes 1 (mTORC1) and 2 (mTORC2). However, recent discoveries have started to unravel the roles of DEPTOR in mTOR-independent responses. In the past few years, mTOR emerged as an important regulator of skeletal development, growth, and homeostasis; the dysregulation of its activity contributes to the development of several skeletal diseases, both chronic and genetic. Even more recently, several groups have reported on the relevance of DEPTOR in skeletal biology through its action on mTORdependent and mTOR-independent pathways. In this review, we summarize the current understanding of DEPTOR in skeletal development and disease.Junta de Andalucia: CV20-81404, UMA18-FEDERJA-177. CIBER Actions, the Instituto de Salud Carlos III; Regional Government of Andalusia (PAIDI group BIO-217); and University of Malaga (Plan Propio).Ye

Two strawberry miR159 family members display developmental-specific expression patterns in the fruit receptacle and cooperatively regulate Fa-GAMYB

We have reported previously that the gibberellin (GA) content in strawberry receptacle is high, peaking at specific stages, pointing to a role of this hormone in fruit development. In Arabidopsis, miR159 levels are dependent on GA concentration. This prompted us to investigate the role of two members of the miR159 family and their putative strawberry target gene, GAMYB, in relation to changes in GA content during the course of fruit development. • The highest expression level of the two Fa-MIR159 genes was in the fruit's receptacle tissue, with dramatic changes observed throughout development. The lowest levels of total mature miR159 (a and b) were observed during the white stage of receptacle development, which was concurrent with the highest expression of Fa-GAMYB. A functional interaction between miR159 and Fa-GAMYB has been demonstrated in receptacle tissue. • The application of bioactive GA (i.e. GA(3) ) to strawberry plants caused the down-regulated expression of Fa-MIR159a, but the expression of Fa-MIR159b was not affected significantly. Clear discrepancies between Fa-MIR159b and mature Fa-miR159b levels were indicative of post-transcriptional regulation of Fa-MIR159b gene expression. • We propose that Fa-miR159a and Fa-miR159b interact with Fa-GAMYB during the course of strawberry receptacle development, and that they act in a cooperative fashion to respond, in part, to changes in GA endogenous levelsThis work was supported by BIO2010-15630 (V.V.) and BIO2009-12004 (C.L.) (Ministerio de Ciencia e Innovacion (MICINN), Spain). F.C. was granted a FPI fellowship from MICINN (Spain). The authors thank Dr Anthony A. Millar for providing Arabidopsis thaliana mir159a, b double-mutant seedsPeer reviewe