Snorc is a novel cartilage specific small membrane proteoglycan expressed in differentiating and articular chondrocytes.

OBJECTIVE: Maintenance of chondrocyte phenotype is a major issue in prevention of degeneration and repair of articular cartilage. Although the critical pathways in chondrocyte maturation and homeostasis have been revealed, the in-depth understanding is deficient and novel modifying components and interaction partners are still likely to be discovered. Our focus in this study was to characterize a novel cartilage specific gene that was identified in mouse limb cartilage during embryonic development. METHODS: Open access bioinformatics tools and databases were used to characterize the gene, predicted protein and orthologs in vertebrate species. Immunohistochemistry and mRNA expression methodology were used to study tissue specific expression. Fracture callus and limb bud micromass culture were utilized to study the effects of BMP-2 during experimental chondrogenesis. Fusion protein with C-terminal HA-tag was expressed in Cos7 cells, and the cell lysate was studied for putative glycosaminoglycan attachment by digestion with chondroitinase ABC and Western blotting. RESULTS: The predicted molecule is a small, 121 amino acids long type I single-pass transmembrane chondroitin sulfate proteoglycan, that contains ER signal peptide, lumenal/extracellular domain with several threonines/serines prone to O-N-acetylgalactosamine modification, and a cytoplasmic tail with a Yin-Yang site prone to phosphorylation or O-N-acetylglucosamine modification. It is highly conserved in mammals with orthologs in all vertebrate subgroups. Cartilage specific expression was highest in proliferating and prehypertrophic zones during development, and in adult articular cartilage, expression was restricted to the uncalcified zone, including chondrocyte clusters in human osteoarthritic cartilage. Studies with experimental chondrogenesis models demonstrated similar expression profiles with Sox9, Acan and Col2a1 and up-regulation by BMP-2. Based on its cartilage specific expression, the molecule was named Snorc, (Small NOvel Rich in Cartilage). CONCLUSION: A novel cartilage specific molecule was identified which marks the differentiating chondrocytes and adult articular chondrocytes with possible functions associated with development and maintenance of chondrocyte phenotype.

Resorption-cycle-dependent polarization of mRNAs for different subunits of V-ATPase in bone-resorbing osteoclasts.

Protein sorting in eukaryotic cells is mainly done by specific targeting of polypeptides. The present evidence from oocytes, neurons, and some other polarized cells suggests that protein sorting can be further facilitated by concentrating mRNAs to their corresponding subcellular areas. However, very little is known about the mechanism(s) involved in mRNA targeting, or how widespread and dynamic such mRNA sorting might be. In this study, we have used an in vitro cell culture system, where large multinucleated osteoclasts undergo continuous structural and functional changes from polarized (resorbing) to a nonpolarized (resting) stage. We demonstrate here, using a nonradioactive in situ hybridization technique and confocal microscopy, that mRNAs for several vacuolar H(+)-ATPase subunits change their localization and polarity in osteoclasts according to the resorption cycle, whereas mRNA for cytoplasmic carbonic anhydrase II is found diffusely located throughout the osteoclast during the whole resorption cycle. Antisense RNA against the 16-kDa or 60-kDa V-ATPase subunit inhibits polarization of the osteoclasts, as determined by cytoskeleton staining. Antisense RNA against carbonic anhydrase II, however, has no such effect.

Do microRNAs regulate bone marrow stem cell niche physiology?

The adult bone marrow, situated within the bone cavity, comprises three distinct stem cell populations: hematopoietic stem cells (HSCs), mesenchymal stromal/stem cells (MSCs) and endothelial progenitor/stem cells (EPCs). HSCs are a well-characterized population of self-renewing cells that give rise to all blood cells. The definition of MSCs is more complex due to the limited understanding of MSC properties. In general, MSCs are considered multipotent stromal cells that are able to differentiate into various cell types, including osteoblasts, chondrocytes and adipocytes. Compared to HSCs and MSCs, EPCs are a newly discovered population of stem/progenitor cells with the capacity to differentiate into endothelial cells, the cells forming the inner lining of a blood vessel. Although functionally different, HSCs, MSCs and EPCs, like stem cells in general, share the ability to self-renew and differentiate into one or more cell types. The homeostasis inside the bone marrow and within the entire body is sustained by an intricate network of growth factors and transcription factors that orchestrate the proliferation and differentiation of these multipotent stem/progenitor cells. Increasing evidence indicates that microRNAs (miRNAs), small non-coding RNAs, are among the key players of this concert. This review summarizes the current insights into miRNA-mediated regulation of bone marrow stem/progenitor cell maintenance and differentiation. Furthermore, the potential contribution of miRNAs in bone marrow stem cell niches is discussed.

Decreased bone resorption, osteoclast differentiation, and expression of vacuolar H+-ATPase in antisense DNA-treated mouse metacarpal and calvaria cultures ex vivo.

Expression and function of vacuolar H(+)-ATPase, a key enzyme in bone resorption, were monitored in antisense DNA-treated bone organ cultures ex vivo. A novel fluoroimmunoassay was used to quantitate mRNA levels after treatment with various antisense, sense, or random DNA oligonucleotides. Conventional slot blots and in vitro translation experiments were used to monitor the efficiency of the antisense molecules. In cell cultures, the used antisense molecules were transported into osteoclasts and a population of mononuclear cells. A significant decrease in bone resorption and in the expression of the 16 kDa, 31 kDa, 42 kDa, 60 kDa, 70 kDa, and 116 kDa subunits of V-ATPase was seen after antisense treatment. Also, osteoclast differentiation was decreased in antisense-treated mouse metacarpal cultures. These data show that the proper function of V-ATPase in osteoclasts requires expression of the 16 kDa, 31 kDa, 42 kDa, 60 kDa, 70 kDa, and 116 kDa subunits of V-ATPase. Antisense DNA molecules can be used to inhibit osteoclast differentiation and function in tissue cultures, in which the physical and chemical cellular environment resembles that in vivo. However, more studies are needed to learn if antisense DNA molecules can be used for inhibiting bone resorption also in vivo.

Downregulation of small GTPase Rab7 impairs osteoclast polarization and bone resorption.

During skeletal growth and remodeling the mineralized bone matrix is resorbed by osteoclasts through the constant secretion of protons and proteases to the bone surface. This relies on the formation of specialized plasma membrane domains, the sealing zone and the ruffled border, and vectorial transportation of intracellular vesicles in bone-resorbing osteoclasts. Here we show that Rab7, a small GTPase that is associated with late endosomes, is highly expressed and is predominantly localized at the ruffled border in bone-resorbing osteoclasts. The decreased expression of Rab7 in cultured osteoclasts by antisense oligodeoxynucleotides disrupted the polarization of the osteoclasts and the targeting of vesicles to the ruffled border. These impairments caused a significant inhibition of bone resorption in vitro. The results indicate that the late endocytotic pathway is involved in the osteoclast polarization and bone resorption and underscore the importance of Rab7 in osteoclast function.

The regulation of pHi in osteoclasts is dependent on the culture substrate and on the stage of the resorption cycle.

The present study was performed to clarify the role of vacuolar H+-ATPase in the regulation of the intracellular pH (pHi) in osteoclasts. Bafilomycin A1 or amiloride were added to rat osteoclast cultures to block the H+-ATPases and Na+/H+-exchangers, respectively. Addition of 10(-8) M bafilomycin A1 to osteoclasts cultured on bone induced a rapid decrease of the pHi, while addition of amiloride had only a minor effect. The response to bafilomycin A1 appeared simultaneously with resorption activity and was abolished when resorption was inhibited by calcitonin. Osteoclasts on bone recovered from acid loading caused by propionate in the presence of amiloride, while bafilomycin A1 inhibited this recovery almost completely. The pHi of osteoclasts cultured on glass responded to the addition of amiloride, but was not effected by even high concentrations of bafilomycin A1. In contrast, as little as 10(-10) M bafilomycin A1 caused accumulation of large vesicles in the cytoplasm.

Carbonic anhydrase II plays a major role in osteoclast differentiation and bone resorption by effecting the steady state intracellular pH and Ca2+.

Carbonic anhydrase II (CA II) expression in characteristic for the early stage of osteoclast differentiation. To study how CA II, which is crucial in proton generation in mature osteoclasts, influences the osteoclast differentiation process we performed rat bone marrow cultures. In this model, acetazolamide, a specific CA inhibitor, decreased the 1,25 (OH)2D3-induced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)-positive cells, in a dose-dependent manner. We then performed intracellular pH (pHi) and Ca2+ (Cai2+) measurements for cultured osteoclasts and noticed that addition of acetazolamide caused a rapid, transient increase of both parameters. The increase in pHi was dependent neither on the culture substrate nor on the extracellular pH (pHe) but the increase could be diminished by DIDS or by bicarbonate removal. Membrane-impermeable CA inhibitors (benzolamide and pd5000) did not have this effect. Addition of CA II antisense oligonucleotides into the cultures reduced the pHi increase significantly. CA II inhibition was also found to neutralize the intracellular vesicles at extracellular pH (pHe) of 7.4, but at less extent at pHe 7.0. In mouse calvaria cultures, bone resorption was inhibited dose dependently by acetazolamide at pHe 7.4 while inhibition was smaller at pHe 7.0. We conclude that CA II is essential not only in bone resorption but also in osteoclast differentiation. In both processes, however, the crucial role of CA II is at least partially due to the effect on the osteoclast pHi regulation.

Inhibition of intravacuolar acidification by antisense RNA decreases osteoclast differentiation and bone resorption in vitro.

The role of proton transport and production in osteoclast differentiation was studied in vitro by inhibiting the transcription/translation of carbonic anhydrase II (CA II) and vacuolar H(+)-ATPase (V-ATPase) by antisense RNA molecules. Antisense RNAs targeted against CA II, or the 16 kDa or 60 kDa subunit of V-ATPase were used to block the expression of the specific proteins. A significant decrease in bone resorption rate and TRAP-positive osteoclast number was seen in rat bone marrow cultures and fetal mouse metacarpal cultures after antisense treatment. Intravacuolar acidification in rat bone marrow cells was also significantly decreased after antisense treatment. The CA II antisense RNA increased the number of TRAP-positive mononuclear cells, suggesting inhibition of osteoclast precursor fusion. Antisense molecules decreased the number of monocytes and macrophages, but increased the number of granulocytes in marrow cultures. GM-CSF, IL-3 and IL-6 were used to stimulate haematopoietic stem cell differentiation. The 16 kDa V-ATPase antisense RNA abolished the stimulatory effect of GM-CSF, IL-3 and IL-6 on TRAP-positive osteoclast formation, but did not affect the formation of monocytes and macrophages after IL-3 treatment, or the formation of granulocytes after IL-6 treatment. These results suggest that CA II and V-ATPase are needed, not only for the actual resorption, but also for osteoclast formation in vitro.