Regulation of secreted Frizzled-related protein-1 by heparin

Secreted Frizzled-related protein-1 (sFRP-1) belongs to a class of extracellular antagonists that modulate Wnt signaling pathways by preventing ligand-receptor interactions among Wnts and Frizzled membrane receptor complexes. sFRP-1 and Wnts are heparin-binding proteins, and their interaction can be stabilized by heparin in vitro. Here we report that heparin can specifically enhance recombinant sFRP-1 accumulation in a cell type-specific manner. The effect requires O-sulfation in heparin, and involves fibroblast growth factor-2 as well as fibroblast growth factor receptor-1. Interestingly, further investigation uncovers that heparin can also affect the post-translational modification of sFRP-1. We demonstrate that sFRP-1 is post-translationally modified by tyrosine sulfation at tyrosines 34 and 36, which is inhibited by the treatment of heparin. The results suggest that accumulation of sFRP-1 induced by heparin is in part due to the relative stabilization of unsulfated sFRP-1 and the direct stabilization by heparin. The study has revealed a multifaceted regulation on sFRP-1 protein by heparin.     

Fairy, tadpole, and clam shrimps (Branchiopoda) in seasonally inundated clay pans in the western Mojave Desert and effect on primary producers

Background

Fairy shrimps (Anostraca), tadpole shrimps (Notostraca), clam shrimps (Spinicaudata), algae (primarily filamentous blue-green algae [cyanobacteria]), and suspended organic particulates are dominant food web components of the seasonally inundated pans and playas of the western Mojave Desert in California. We examined the extent to which these branchiopods controlled algal abundance and species composition in clay pans between Rosamond and Rogers Dry Lakes. We surveyed branchiopods during the wet season to estimate abundances and then conducted a laboratory microcosm experiment, in which dried sediment containing cysts and the overlying algal crust were inundated and cultured. Microcosm trials were run with and without shrimps; each type of trial was run for two lengths of time: 30 and 60 days. We estimated the effect of shrimps on algae by measuring chlorophyll content and the relative abundance of algal species.

Results

We found two species of fairy shrimps (Branchinecta mackini and B. gigas), one tadpole shrimp (Lepidurus lemmoni), and a clam shrimp (Cyzicus setosa) in our wet-season field survey. We collected Branchinecta lindahli in a pilot study, but not subsequently. The dominant taxa were C. setosa and B. mackini, but abundances and species composition varied greatly among playas. The same species found in field surveys also occurred in the microcosm experiment. There were no significant differences as a function of experimental treatments for either chlorophyll content or algal species composition (Microcoleus vaginatus dominated all treatments).

Conclusions

The results suggest that there was no direct effect of shrimps on algae. Although the pans harbored an apparently high abundance of branchiopods, these animals had little role in regulating primary producers in this environment.     

Wnt signaling control of bone cell apoptosis

Wnts are a large family of growth factors that mediate essential biological processes like embryogenesis, morpho- genesis and organogenesis. These proteins also play a role in oncogenesis, and they regulate apoptosis in many tissues. Wnts bind to a membrane receptor complex comprised of a frizzled （FZD） G-protein-coupled receptor and a low-density lipoprotein （LDL） receptor-related protein （LRP）. The formation of this ligand-receptor complex initiates a number of signaling cascades that include the canonical/beta-catenin pathway as well as several noncanonical pathways. In recent years, canonical Wnt signaling has been reported to play a significant role in the control of bone formation. Clinical studies have found that mutations in LRP-5 are associated with reduced bone mineral density （BMD） and fractures. Investigations of knockout and transgenic mouse models of Wnt pathway components have shown that canonical Wnt signaling modulates most aspects ofosteoblast physiology including proliferation, differentiation, function and apoptosis. Transgenic mice expressing a gain of function mutant of LRP-5 in bone, or mice lacking the Wnt antagonist secreted frizzled-related protein-l, exhibit elevated BMD and suppressed osteoblast apoptosis. In addition, preclinical studies with pharmacologic compounds such as those that inhibit glycogen synthase kinase-3β support the importance of the canonical Wnt pathway in modulation of bone formation and osteoblast apoptosis.     

The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis

Mechanisms controlling human bone formation remain to be fully elucidated. We have used differential display-polymerase chain reaction analysis to characterize osteogenic pathways in conditionally immortalized human osteoblasts (HOBs) representing distinct stages of differentiation. We identified 82 differentially expressed messages and found that the Wnt antagonist secreted frizzled-related protein (sFRP)-1 was the most highly regulated of these. Transient transfection of HOBs with sFRP-1 suppressed canonical Wnt signaling by 70% confirming its antagonistic function in these cells. Basal sFRP-1 mRNA levels increased 24-fold during HOB differentiation from pre-osteoblasts to pre-osteocytes, and then declined in mature osteocytes. This expression pattern correlated with levels of cellular viability such that the pre-osteocytes, which had the highest levels of sFRP-1 mRNA, also had the highest rate of cell death. Basal sFRP-1 mRNA levels also increased 29-fold when primary human mesenchymal stem cells were differentiated to osteoblasts supporting the developmental regulation of the gene. Expression of sFRP-1 mRNA was induced 38-fold following prostaglandin E2 (PGE2) treatment of pre-osteoblasts and mature osteoblasts that had low basal message levels. In contrast, sFRP-1 expression was down-regulated by as much as 80% following transforming growth factor (TGF)-beta1 treatment of pre-osteocytes that had high basal mRNA levels. Consistent with this, treatment of pre-osteoblasts and mature osteoblasts with PGE(2) increased apoptosis threefold, while treatment of pre-osteocytes with TGF-beta1 decreased cell death by 50%. Likewise, over-expression of sFRP-1 in HOBs accelerated the rate of cell death threefold. These results establish sFRP-1 as an important negative regulator of human osteoblast and osteocyte survival.     

GATA-1 and Oct-1 are required for expression of the human alpha-hemoglobin-stabilizing protein gene

Alpha-hemoglobin-stabilizing protein (AHSP) is an erythroid protein that binds and stabilizes alpha-hemoglobin during normal erythropoiesis and in pathological states of alpha-hemoglobin excess. AHSP has been proposed as a candidate gene in some Heinz body hemolytic anemias and as a modifier gene in the beta-thalassemia syndromes. To gain additional insight into the molecular mechanisms controlling the erythroid-specific expression of the AHSP gene and provide the necessary tools for further genetic studies of these disorders, we have initiated identification and characterization of the regulatory elements controlling the human AHSP gene. We mapped the 5'-end of the AHSP erythroid cDNA and cloned the 5'-flanking genomic DNA containing the putative AHSP gene promoter. In vitro studies using transfection of promoter/reporter plasmids in human tissue culture cell lines, DNase I footprinting analyses and gel mobility shift assays, identified an AHSP gene erythroid promoter with functionally important binding sites for GATA-1- and Oct-1-related proteins. In transgenic mice, a reporter gene directed by a minimal human AHSP promoter was expressed in bone marrow, spleen, and reticulocytes, but not in nonerythroid tissues. In vivo studies using chromatin immunoprecipitation assays demonstrated hyperacetylation of the promoter region and occupancy by GATA-1. The AHSP promoter is an excellent candidate region for mutations associated with decreased AHSP gene expression.     

A 46 base pair enhancer sequence within the locus activating region is required for induced expression of the gamma-globin gene during erythroid differentiation.

The locus activating region (LAR), contained within 30 kb of chromatin flanking the human beta-globin gene cluster, has recently been shown to be essential for high level beta-globin gene expression. To determine the effect of fragments containing LAR sequences on globin gene expression, mRNA from a marked gamma-globin gene linked to LAR fragments was assayed in stably transfected K562 erythroleukemia cells. DNaseI hypersensitive site II (HS II), located 10.9 kb upstream of the epsilon-globin gene, was required for high level gamma-globin gene expression. We also showed that a 46 bp enhancer element within HS II was necessary and sufficient for the increased gamma-globin gene expression observed with hemin induced erythroid maturation of K562 cells. These results localize a distant regulatory element important for activation of globin genes during human erythroid cell maturation.     

PTH-dependent adenylyl cyclase activation in SaOS-2 cells: passage dependent effects on G protein interactions

Parathyroid hormone (PTH) sensitive adenylyl cyclase activity (ACA) in SaOS-2 cells varies as a function of cell passage. In early passage (EP) cells (< 6), ACA in response to PTH and forskolin (FOR) was relatively low and equivalent, whereas in late passage (LP) cells (> 22), PTH exceeded FOR dependent ACA. Potential biochemical mechanisms for this passage dependent change in ACA were considered. In EP, prolonged exposure to pertussis toxin (PT) markedly enhanced ACA activity in response to PTH, Isoproterenol and Gpp(NH)p, whereas ACA in response to FOR was decreased. In contrast, the identical treatment of LP with PT diminished all ACA in response to PTH, Gpp(NH)p, and FOR. The dose dependent effects of PT on subsequent [(32)P]ADP-ribosylation of its substrates, GTPase activity, as well as FOR-dependent ACA, were equivalent in EP and LP. The relative amounts of G(alpha)i and G(alpha)s proteins, as determined both by Western blot, PT and cholera toxin (CT) dependent [(32)P]ADP-ribosylation, were quantitatively similar in EP and LP. Western blot levels of G(alpha)s and G(alpha)i proteins were not influenced by prior exposure to PT. Both PT and CT dependent [(32)P]ADP-ribosylation were dose-dependently decreased following exposure to PT. However, the PT-dependent decline in CT-dependent [(32)P]ADP-ribosylation occurred with enhanced sensitivity in LP. The protein synthesis inhibitor cycloheximide partially reversed the PT associated decrease in FOR dependent ACA in EP. In contrast, cycloheximide completely reversed the PT associated decrease in FOR and as well as PTH dependent ACA in LP. G(alpha)s activity, revealed by cyc(-) reconstitution, was not altered either by cell passage or exposure to PT. The results suggest that the coupling between the components of the complex may be pivotally important in the differential responsiveness of early and late passage SaOS-2 cells to PTH.     

Systemically administered rhBMP-2 promotes MSC activity and reverses bone and cartilage loss in osteopenic mice

Osteoporosis is a disease manifested in drastic bone loss resulting in osteopenia and high risk for fractures. This disease is generally divided into two subtypes. The first, post-menopausal (type I) osteoporosis, is primarily related to estrogen deficiency. The second, senile (type II) osteoporosis, is mostly related to aging. Decreased bone formation, as well as increased bone resorption and turnover, are thought to play roles in the pathophysiology of both types of osteoporosis. In this study, we demonstrate in murine models for both type I (estrogen deficiency) and type II (senile) osteopenia/osteoporosis that reduced bone formation is related to a decrease in adult mesenchymal stem cell (AMSC) number, osteogenic activity, and proliferation. Decreased proliferation is coupled with increased apoptosis in AMSC cultures obtained from osteopenic mice. Recombinant human bone morphogenetic protein (rhBMP-2) is a highly osteoinductive protein, promoting osteogenic differentiation of AMSCs. Systemic intra-peritoneal (i.p.) injections of rhBMP-2 into osteopenic mice were able to reverse this phenotype in the bones of these animals. Moreover, this change in bone mass was coupled to an increase in AMSCs numbers, osteogenic activity, and proliferation as well as a decrease in apoptosis. Bone formation activity was increased as well. However, the magnitude of this response to rhBMP-2 varied among different stains of mice. In old osteopenic BALB/c male mice (type II osteoporosis model), rhBMP-2 systemic treatment also restored both articular and epiphyseal cartilage width to the levels seen in young mice. In summary, our study shows that AMSCs are a good target for systemically active anabolic compounds like rhBMP-2.