Expression of multiple insulin and insulin-like growth factor receptor genes in salmon gill cartilage

In mammals, one of the major actions of insulin-like growth factor I (IGF-I) is to increase skeletal growth by stimulating new cartilage formation. IGF-I stimulates chondrocytes in vitro to synthesize new cartilage matrix, measured by enhanced uptake of 35S-sulfate, but the addition of insulin does not produce a similar effect except when added at high concentrations. However, recent studies have shown that, in teleosts, both insulin and IGF-I are potent activators of 35S-sulfate uptake in gill cartilage. To further characterize the growth-promoting activities of these hormones in fish, we have used reverse transcriptase-linked PCR to analyze the expression of insulin receptor family genes in salmon gill cartilage. Partial cDNA sequences encoding the tyrosine kinase domains from six distinct members of the IR gene family were obtained, and sequence comparisons revealed that four of the cDNAs encoded amino acid sequences that were highly homologous to human IR whereas the encoded sequences from two of the cDNAs were more similar to the human type I IGF receptor (IGF-R). Furthermore, a comparative reverse transcriptase-linked PCR assay revealed that the four putative IR mRNAs expressed in toto in gill cartilage were 56% of that found in liver whereas the expressed amount of the two IGF-R mRNAs was 9-fold higher compared with liver. These results suggest that the chondrogenic actions of insulin and IGF-I in fish are mediated by the ligands binding to their cognate receptors. However, further studies will be required to characterize the binding properties and relative contribution of the individual IR and IGF-R genes.

Bone marrow-derived mesenchymal stem cells become anti-angiogenic when chondrogenically or osteogenically differentiated:implications for bone and cartilage tissue engineering

Osteochondral tissue repair requires formation of vascularized bone and avascular cartilage. Mesenchymal stem cells stimulate angiogenesis both in vitro and in vivo but it is not known if these proangiogenic properties change as a result of chondrogenic or osteogenic differentiation. We investigated the angiogenic/antiangiogenic properties of equine bone marrow-derived mesenchymal stem cells (eBMSCs) before and after differentiation in vitro. Conditioned media from chondrogenic and osteogenic cell pellets and undifferentiated cells was applied to endothelial tube formation assays using Matrigel™. Additionally, the cell secretome was analysed using LC-MS/MS mass spectrometry and screened for angiogenesis and neurogenesis-related factors using protein arrays. Endothelial tube-like formation was supported by conditioned media from undifferentiated eBMSCs. Conversely, chondrogenic and osteogenic conditioned media was antiangiogenic as shown by significantly decreased length of endothelial tube-like structures and degree of branching compared to controls. Undifferentiated cells produced higher levels of angiogenesis-related proteins compared to chondrogenic and osteogenic pellets. In summary, eBMSCs produce an array of angiogenesis-related proteins and support angiogenesis in vitro via a paracrine mechanism. However, when these cells are differentiated chondrogenically or osteogenically, they produce a soluble factor(s) that inhibits angiogenesis. With respect to osteochondral tissue engineering, this may be beneficial for avascular articular cartilage formation but unfavourable for bone formation where a vascularized tissue is desired. © Copyright 2014, Mary Ann Liebert, Inc.

Isolation and in vitro chondrogenic potential of human foetal spine cells.

Cell therapy for nucleus pulposus (NP) regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous NP cells or stem cells. Another potential source of cells is foetal cells. We investigated the feasibility of isolating foetal cells from human foetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of foetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of foetal intervertebral disc (IVD) were similar to adult IVD. Isolated foetal cells were cultured in monolayer in basic media supplemented with 10% Fetal Bovine Serum (FBS) and from each foetal tissue donation, a cell bank of foetal spine cells at passage 2 was established and was composed of around 2000 vials of 5 million cells. Gene expression and immunohistochemistry of foetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, foetal cells could be isolated from human foetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for IVD regeneration.

Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

Morphological deformities in the osseous structure in spotted sorubim Pseudoplatystoma coruscans (agassiz & spix, 1829) with vitamin c deficiency

A vitamina C é essencial para dietas de peixe porque muitas espécies não conseguem sintetizá-la. Esta vitamina é necessária par a formação de cartilagem e matriz óssea. Além disso, age como antioxidante e melhora as resposta do sistema imunológico. O presente trabalho investigou os efeitos da suplementação de vitamina C em dietas para alevinos de pintado (Pseudoplatystoma corruscans) pela incidência de deformidades na estrutura óssea e cartilaginosa. O ascorbil polifosfato (AP) foi utilizado como fonte de vitamina C em dietas para alevinos de pintado durante o período de três meses. Seis dietas foram formuladas: uma dieta controle (0 mg de vitamina C / kg) e cinco dietas 500, 1.000, 1.500, 2.000 e 2.500 mg de AP / kg. Os peixes alimentados sem suplementação de vitamina C apresentaram deformidades óssea na cabeça e mandíbula e fragilidade de nadadeiras. Assim, a dieta de 500 mg de AP/kg foi suficiente para prevenir a ocorrência de deformidades, e a ausência desta vitamina prejudica o desenvolvimento ósseo de juvenis de pintados.

Cultivo de condrócitos equinos e avaliação de sua condrogenicidade mediante implante de células associadas ao hidrogel em subcutâneo de camundongos atímicos

Pós-graduação em Medicina Veterinária - FMVZ

Avaliação do laser de baixa intensidade na regeneração de cartilagem articular do joelho de coelhos submetidos à trocleoplastia

Pós-graduação em Cirurgia Veterinária - FCAV

Morphological deformities in the osseous structure in spotted sorubim Pseudoplatystoma coruscans (agassiz & spix, 1829) with vitamin c deficiency

A vitamina C é essencial para dietas de peixe porque muitas espécies não conseguem sintetizá-la. Esta vitamina é necessária par a formação de cartilagem e matriz óssea. Além disso, age como antioxidante e melhora as resposta do sistema imunológico. O presente trabalho investigou os efeitos da suplementação de vitamina C em dietas para alevinos de pintado (Pseudoplatystoma corruscans) pela incidência de deformidades na estrutura óssea e cartilaginosa. O ascorbil polifosfato (AP) foi utilizado como fonte de vitamina C em dietas para alevinos de pintado durante o período de três meses. Seis dietas foram formuladas: uma dieta controle (0 mg de vitamina C / kg) e cinco dietas 500, 1.000, 1.500, 2.000 e 2.500 mg de AP / kg. Os peixes alimentados sem suplementação de vitamina C apresentaram deformidades óssea na cabeça e mandíbula e fragilidade de nadadeiras. Assim, a dieta de 500 mg de AP/kg foi suficiente para prevenir a ocorrência de deformidades, e a ausência desta vitamina prejudica o desenvolvimento ósseo de juvenis de pintados.

Bone Morphogenetic Proteins: Structure, biological function and therapeutic applications

Bone Morphogenetic Proteins (BMPs) are multifunctional secreted cytokines, which belong to the TGF-beta superfamily. These glycoproteins act as a disulfide-linked homo- or heterodimers, being potent regulators of bone and cartilage formation and repair, cell proliferation during embryonic development and bone homeostasis in the adult. BMPs are promising molecules for tissue engineering and bone therapy. The present review discusses this family of proteins, their structure and biological function, their therapeutic applications and drawbacks, their effects on mesenchymal stem cells differentiation, and the cell signaling pathways involved in this process. (C) 2014 Published by Elsevier Inc.

Regulations of SOX9 activity during chondrogenesis

Sox9 is a transcription factor required for chondrocyte differentiation and cartilage formation. In an effort to identify SOX9 interacting protein(s), we screened a chondrocyte cDNA library with a modified yeast two-hybrid method, Son of Sevenless (SOS) recruitment system (SRS). The catalytic subunit of cyclic AMP-dependent protein kinase A (PKA-Cα) and a new long form of c-Maf transcription factor (Lc-Maf) were found to interact specifically with SOX9. We showed here that two PKA phosphorylation consensus sites of SOX9 could be phosphorylated by PKA in vitro as well as in vivo. PKA phosphorylation of SOX9 increases its DNA binding and transcriptional activities on a Col2a1 chondrocyte-specific enhancer. Mutations of these two PKA phosphorylation sites markedly decreased the activation of SOX9 by PKA. ^ To test whether parathyroid hormone-related peptide (PTHrP) signaling results in SOX9 phosphorylation, we generated a phosphospecific antibody that specifically recognizes SOX9 that is phosphorylated at serine 181 (S 181) one of the two consensus PKA phosphorylation sites. Addition of PTHrP to COS7 cells cotransfected with SOX9 and PTH/PTHrP receptor strongly increased phosphorylation of SOX9 at S181; this phosphorylation was blocked by a PKA-specific inhibitor. In similar experiments we showed that PTHrP increased the activity of a SOX9-dependent Col2a1 enhancer. This increase in activity was abolished when a SOX9 mutant was used containing serine-to-alanine substitution in the two consensus PKA phosphorylation sites of SOX9. Using our phosphospecific SOX9 antibody we showed by immunohistochemistry of mouse embryos that Sox9 phosphorylated at S181 was localized almost exclusively in the pre-hypertrophic zone of the growth plate, an area corresponding to the major site of expression of PTH/PTHrP receptor. In contrast, no phosphorylation of Sox9 at S181 was detected in growth plates of PTH/PTHrP receptor null mutant mice. Sox9, regardless of phosphorylation state, was present in all chondrocytes of both genotypes except in hypertrophic chondrocytes. Thus, Sox9 is a target of PTHrP signaling and the PTHrP-dependent phosphorylation of SOX9 enhances its transcriptional activity. ^ In order to investigate the in vivo function of Sox9 phosphorylation by PKA, we are generating a mouse model of mutant Sox9 harboring point mutations in two PKA phosphorylation sites. Preliminary results indicated that heterozygous mice containing half amount of mutant Sox9 that can not be phosphorylated by PKA have normal skeletal phenotype and homozygous mice are being generated. ^ Lc-Maf encodes an extra ten amino acids at the carboxyl terminus of c-Maf and contains a completely different 3′ untranslated region. The interaction between SOX9 and Lc-Maf was further confirmed by co-immunoprecipitation and GST-pull down assays, which mapped the interacting domains of SOX9 to HMG DNA binding domain and that of Lc-Maf to basic leusine zipper motif. In situ hybridizations showed that RNA of Lc-Maf coexpressed with those of Sox9 and Col2a1 in areas of mesenchymal condensation during the early stages of mouse embryo development. A DNA binding site of Lc-Maf was identified at the 5′ part of a 48-bp Col2a1 enhancer element near the HMG binding site of SOX9. Lc-Maf and SOX9 synergistically activated a luciferase reporter plasmid containing a Col2al enhancer and increased the transcription of endogenous Col2a1 gene. In summary, Lc-Maf is the first identified SOX9-interating protein during chondrogenesis and may be an important activator of Col2a1 gene. ^

Characterization of the human sulfate anion transporter (hsat-1) protein and gene (SAT1; SLC26A1)

Sulfate plays an essential role during growth, development, bone/cartilage formation, and cellular metabolism. In this study, we have isolated the human sulfate anion transporter cDNA (hsat-1; SCL26A1) and gene (SAT1), determined its protein function in Xenopus oocytes and characterized SAT1 promoter activity in mammalian renal cell lines. hsat-1 encodes a protein of 75 kDa, with 12 putative transmembrane domains, that induces sulfate, chloride, and oxalate transport in Xenopus oocytes. hsat-1 mRNA is expressed most abundantly in the kidney and liver, with lower levels in the pancreas, testis, brain, small intestine, colon, and lung. The SAT1 gene is comprised of four exons stretching 6 kb in length, with an alternative splice site formed from an optional exon. SAT1 5' flanking region led to promoter activity in renal OK and LLC-PK1 cells. Using SAT1 5' flanking region truncations, the first 135 bp was shown to be sufficient for basal promoter activity. Mutation of the activator protein-1 (AP-1) site at position 252 in the SAT1 promoter led to loss of transcriptional activity, suggesting its requirement for SAT1 basal expression. This study represents the first functional characterization of the human SAT1 gene and protein encoded by the anion transporter hsat-1.

The mouse sulfate anion transporter gene Sat1 (Slc26a1): Cloning, tissue distribution, gene structure, functional characterization, and transcriptional regulation by thyroid hormone

Sulfate (SO42-) is required for bone/cartilage formation and cellular metabolism. sat-1 is a SO42- anion transporter expressed on basolateral membranes of renal proximal tubules, and is suggested to play an important role in maintaining SO42- homeostasis. As a first step towards studying its tissue-specific expression, hormonal regulation, and in preparation for the generation of knockout mice, we have cloned and characterized the mouse sat-1 cDNA (msat-1), gene (sat1; Slc26a1) and promoter region. msat-1 encodes a 704 amino acid protein (75.4 kDa) with 12 putative transmembrane domains that induce SO42- (also oxalate and chloride) transport in Xenopus oocytes. msat-1 mRNA was expressed in kidney, liver, cecum, calvaria, brain, heart, and skeletal muscle. Two distinct transcripts were expressed in kidney and liver due to alternative utilization of the first intron, corresponding to an internal portion of the 5'-untranslated region. The Sa1 gene (similar to6 kb) consists of 4 exons. Its promoter is similar to52% G+C rich and contains a number of well-characterized cis-acting elements, including sequences resembling hormone responsive elements T3REs and VDREs. We demonstrate that Sat1 promoter driven basal transcription in OK cells was stimulated by tri-iodothyronine. Site-directed mutagenesis identified an imperfect T3RE at -454-bp in the Sat1 promoter to be responsible for this activity. This study represents the first characterization of the structure and regulation of the Sat1 gene encoding a SO42-/chloride/oxalate anion transporter.

Sex determination: a tale of two Sox genes

Vertebrates use many different strategies to determine sex, but the Sox9 gene is a common thread, probably acting as the pivotal gene that controls the male-determining pathway. It now appears that Sox9 is not alone in this role, and that a closely related gene, Sox8, can partly substitute for Sox9. But is this a clever backup strategy to safeguard male development, or a relic of the past?

Regulation of Amh during sex determination in chickens: Sox gene expression in male and female gonads

During mammalian sexual development, the SOX9 transcription factor up-regulates expression of the gene encoding anti-Mullerian hormone (AMH), but in chickens, Sox9 gene expression reportedly occurs after the onset of Amh expression. Here, we examined expression of the related gene Sox8 in chicken embryonic gonads during the sex-determining period. We found that cSox8 is expressed at similar levels in both sexes at embryonic day 6 and 7, and only at the anterior tip of the gonad, suggesting that SOX8 is not responsible for the sex-specific increase in cAmh gene expression at these stages. We also found that several other chicken Sox genes (cSox3, cSox4 and cSox11) are expressed in embryonic gonads, but at similar levels in both sexes. Our data suggest that the molecular mechanisms involved in the regulation of Amh genes of mouse and chicken are not conserved, despite similar patterns of Amh expression in both species.

Origin and possible roles of the Sox8 transcription factor gene during sexual development

Sox8 is a member of the Sox family of developmental transcription factor genes and is closely related to Sox9, a critical gene involved in mammalian sex determination and differentiation. Both genes encode proteins with the ability to bind similar DNA target sequences, and to activate transcription in in vitro assays. Expression studies indicate that the two genes have largely overlapping patterns of activity during mammalian embryonic development. A knockout of Sox8 in mice has no obvious developmental phenotype, suggesting that the two genes are able to act redundantly in a variety of developmental contexts. In particular, both genes are expressed in the developing Sertoli cell lineage of the developing testes in mice, and both proteins are able to activate transcription of the gene encoding anti-Mullerian hormone (AMH), through synergistic action with steroidogenic factor I (SF1). We have hypothesized that Sox8 may substitute for Sox9 in species where Sox9 is expressed too late to be involved in sex determination or regulation of Amh expression. However, our studies involving the red-eared slider turtle indicate that Sox8 is expressed at similar levels in males and females throughout the sex-determining period, suggesting that Sox8 is neither a transcriptional regulator for Amh, nor responsible for sex determination or gonad differentiation in that species. Similarly, Sox8 is not expressed in a sexually dimorphic pattern during gonadogenesis in the chicken. Since a functional role(s) for Sox8 is implied by its conservation during evolution, the significance of Sox8 for sexual and other aspects of development will need to be uncovered through more directed lines of experimentation. Copyright (C) 2003 S. Karger AG, Basel.