Chondrocytes respond to an altered heparan sulfate composition with distinct changes of heparan sulfate structure and increased levels of chondroitin sulfate.

Heparan sulfate (HS) regulates the activity of many signaling molecules critical for the development of endochondral bones. Even so, mice with a genetically altered HS metabolism display a relatively mild skeletal phenotype compared to the defects observed in other tissues and organs pointing to a reduced HS dependency of growth-factor signaling in chondrocytes. To understand this difference, we have investigated the glycosaminoglycan (GAG) composition in two mouse lines that produce either reduced levels of HS (Ext1gt/gt mice) or HS lacking 2-O-sulfation (Hs2st1-/- mice). Analysis by RPIP-HPLC revealed an increased level of sulfated disaccarides not affected by the mutation in both mouse lines indicating that chondrocytes attempt to restore a critical level of sulfation. In addition, in both mutant lines we also detected significantly elevated levels of CS. Size exclusion chromatography further demonstrated that Ext1gt/gt mutants produce more but shorter CS chains, while the CS chains produced by (Hs2st1-/- mice) mutants are of similar length to that of wild type littermates indicating that chondrocytes produce more rather than longer CS chains. Expression analysis revealed an upregulation of aggrecan, which likely carries most of the additionally produced CS. Together the results of this study demonstrate for the first time that not only a reduced HS synthesis but also an altered HS structure leads to increased levels of CS in mammalian tissues. Furthermore, as chondrocytes produce 100-fold more CS than HS the increased CS levels point to an active, precursor-independent mechanism that senses the quality of HS in a vast excess of CS. Interestingly, reducing the level of cell surface CS by chondroitinase treatment leads to reduced Bmp2 induced Smad1/5/9 phosphorylation. In addition, Erk phosphorylation is increased independent of Fgf18 treatment indicating that both, HS and CS, affect growth factor signaling in chondrocytes in distinct manners.

Altered heparan sulfate structure in Glce(-/-) mice leads to increased Hedgehog signaling in endochondral bones.

One of the key regulators of endochondral ossification is Indian hedgehog (Ihh), which acts as a long-range morphogen in the developing skeletal elements. Previous studies have shown that the distribution and signaling activity of Ihh is regulated by the concentration of the extracellular glycosaminoglycan heparan sulfate (HS). An essential step during biosynthesis of HS is the epimerization of D-glucuronic to L-iduronic acid by the enzyme glucuronyl C5-epimerase (Hsepi or Glce). Here we have investigated chondrocyte differentiation in Glce deficient mice and found increased regions of proliferating chondrocytes accompanied by a delayed onset of hypertrophic differentiation. In addition, we observed increased expression levels of the Ihh target genes Patched1 (Ptch1) and Parathyroid hormone related peptide (Pthrp; Parathyroid hormone like hormone (Pthlh)) indicating elevated Ihh signaling. We further show that Ihh binds with reduced affinity to HS isolated from Glce(-/-) mice. Together our results strongly indicate that not only the level, but also the structure of HS is critical in regulating the distribution and signaling activity of Ihh in chondrocytes.

Sweet on Hedgehogs: regulatory roles of heparan sulfate proteoglycans in Hedgehog-dependent cell proliferation and differentiation.

Morphogens exert their effects over long distances, typically by spreading from cell to cell to activate signal transduction in surrounding tissues in concentration-dependent manner. One example of a morphogen is the signaling molecule Hedgehog (Hh), which controls growth and patterning during development and has also been implicated in the progression of numerous cancers. To this end, accessory mechanisms that release, transport, and receive Hhs are required to elicit temporally and spatially specific responses in cells and tissues. The Hh spreading mechanism is especially intriguing, because all Hhs are released from the producing cells despite being synthesized as dually lipidated, membrane-tethered molecules. In addition to this cellular association, Hhs bind strongly to extracellular heparan sulfate proteoglycans (HSPGs), which is expected to further reduce their spreading. Paradoxically, several lines of evidence suggest that Hh gradient formation actually requires HSPG expression, and that HSPGs act as both positive and negative regulators of Hh function. This article reviews the multiple roles that HSPGs play in Hh morphogen function, and discusses their congruity with proposed mechanisms of Hh solubilization, transport, and signal reception in vertebrate and invertebrate tissues.

Phylogenetic diversity of archaea and the archaeal ammonia monooxygenase gene in uranium mining-impacted locations in Bulgaria.

Uranium mining and milling activities adversely affect the microbial populations of impacted sites. The negative effects of uranium on soil bacteria and fungi are well studied, but little is known about the effects of radionuclides and heavy metals on archaea. The composition and diversity of archaeal communities inhabiting the waste pile of the Sliven uranium mine and the soil of the Buhovo uranium mine were investigated using 16S rRNA gene retrieval. A total of 355 archaeal clones were selected, and their 16S rDNA inserts were analysed by restriction fragment length polymorphism (RFLP) discriminating 14 different RFLP types. All evaluated archaeal 16S rRNA gene sequences belong to the 1.1b/Nitrososphaera cluster of Crenarchaeota. The composition of the archaeal community is distinct for each site of interest and dependent on environmental characteristics, including pollution levels. Since the members of 1.1b/Nitrososphaera cluster have been implicated in the nitrogen cycle, the archaeal communities from these sites were probed for the presence of the ammonia monooxygenase gene (amoA). Our data indicate that amoA gene sequences are distributed in a similar manner as in Crenarchaeota, suggesting that archaeal nitrification processes in uranium mining-impacted locations are under the control of the same key factors controlling archaeal diversity.

Reprint of: Heparan sulfate as a regulator of endochondral ossification and osteochondroma development.

Most elements of the vertebrate skeleton are formed by endochondral ossification. This process is initiated with mesenchymal cells that condense and differentiate into chondrocytes. These undergo several steps of differentiation from proliferating into hypertrophic chondrocytes, which are subsequently replaced by bone. Chondrocyte proliferation and differentiation are tightly controlled by a complex network of signaling molecules. During recent years, it has become increasingly clear that heparan sulfate (HS) carrying proteoglycans play a critical role in controlling the distribution and activity of these secreted factors. In this review we summarize the current understanding of the role of HS in regulating bone formation. In human, mutations in the HS synthetizing enzymes Ext1 and Ext2 induce the Multiple Osteochondroma syndrome, a skeletal disorder characterized by short stature and the formation of benign cartilage-capped tumors. We review the current insight into the origin of the disease and discuss its possible molecular basis. In addition, we summarize the existing insight into the role of HS as a regulator of signal propagation and signaling strength in the developing skeleton.

Heparan sulfate as a regulator of endochondral ossification and osteochondroma development.

Most elements of the vertebrate skeleton are formed by endochondral ossification. This process is initiated with mesenchymal cells that condense and differentiate into chondrocytes. These undergo several steps of differentiation from proliferating into hypertrophic chondrocytes, which are subsequently replaced by bone. Chondrocyte proliferation and differentiation are tightly controlled by a complex network of signaling molecules. During recent years, it has become increasingly clear that heparan sulfate (HS) carrying proteoglycans play a critical role in controlling the distribution and activity of these secreted factors. In this review we summarize the current understanding of the role of HS in regulating bone formation. In human, mutations in the HS synthetizing enzymes Ext1 and Ext2 induce the Multiple Osteochondroma syndrome, a skeletal disorder characterized by short stature and the formation of benign cartilage-capped tumors. We review the current insight into the origin of the disease and discuss its possible molecular basis. In addition, we summarize the existing insight into the role of HS as a regulator of signal propagation and signaling strength in the developing skeleton.

The U(VI) speciation influenced by a novel Paenibacillus isolate from Mont Terri Opalinus clay.

Bacterial cell walls have a high density of ionizable functional groups available for U(VI) binding, hence have a great potential to affect the speciation of this contaminant in the environment. The studied strain of the genus Paenibacillus is a novel isolate originating from the Mont Terri Opalinus clay formations (Switzerland) which are currently investigated as a potential host rock for future nuclear waste storage. U(VI) binding to the cell surface functional groups was studied by potentiometry combined with time-resolved laser-induced fluorescence spectroscopy (TRLFS). Four bacterial U(VI) surface complexes were identified: R-COO-UO2(+), R-O-PO3-UO2, R-O-PO3H-UO2(+), and (R-O-PO3)2-UO2(2-). The corresponding complex stability constants were calculated to be 5.33 ± 0.08, 8.89 ± 0.04, 12.92 ± 0.05, and 13.62 ± 0.08, respectively. Hence UO2(2+) displays a moderate to strong interaction with the bacterial surface functional groups. In the acidic pH range (pH 3) UO2(2+) binding onto the cell envelope is governed by coordination to hydrogen phosphoryl sites. Upon increasing the pH an increasing coordination of UO2(2+) to carboxylic and deprotonated phosphoryl sites was found. At a pH greater than 7 uranyl hydroxides dominate the speciation. Additionally the bacteria-mediated release of inorganic phosphate in dependence on [U(VI)] at different pH values was studied to assess the influence of phosphate release on U(VI) mobilization.