Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Anteroposterior polarity in the vertebrate limb is thought to be regulated in response to signals derived from a specialized region of distal posterior mesenchyme, the zone of polarizing activity. Sonic Hedgehog (Shh) is expressed in the zone of polarizing activity and appears to mediate the action of the zone of polarizing activity. Here we have manipulated Shh signal in the limb to assess whether it acts as a long-range signal to directly pattern all the digits. Firstly, we demonstrate that alterations in digit development are dependent upon the dose of Shh applied. DiI-labeling experiments indicate that cells giving rise to the extra digits lie within a 300 microm radius of a Shh bead and that the most posterior digits come from cells that lie very close to the bead. A response to Shh involves a 12-16 hour period in which no irreversible changes in digit pattern occur. Increasing the time of exposure to Shh leads to specification of additional digits, firstly digit 2, then 3, then 4. Cell marking experiments demonstrate that cells giving rise to posterior digits are first specified as anterior digits and later adopt a more posterior character. To monitor the direct range of Shh signalling, we developed sensitive assays for localizing Shh by attaching alkaline phosphatase to Shh and introducing cells expressing these forms into the limb bud. These experiments demonstrate that long-range diffusion across the anteroposterior axis of the limb is possible. However, despite a dramatic difference in their diffusibility in the limb mesenchyme, the two forms of alkaline phosphatase-tagged Shh proteins share similar polarizing activity. Moreover, Shh-N (aminoterminal peptide of Shh)-coated beads and Shh-expressing cells also exhibit similar patterning activity despite a significant difference in the diffusibility of Shh from these two sources. Finally, we demonstrate that when Shh-N is attached to an integral membrane protein, cells transfected with this anchored signal also induce mirror-image pattern duplications in a dose-dependent fashion similar to the zone of polarizing activity itself. These data suggest that it is unlikely that Shh itself signals digit formation at a distance. Beads soaked in Shh-N do not induce Shh in anterior limb mesenchyme ruling out direct propagation of a Shh signal. However, Shh induces dose-dependent expression of Bmp genes in anterior mesenchyme at the start of the promotion phase. Taken together, these results argue that the dose-dependent effects of Shh in the regulation of anteroposterior pattern in the limb may be mediated by some other signal(s). BMPs are plausible candidates.
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PMID:Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb. 933 87

Members of the vertebrate hedgehog gene family (HH) are involved in patterning and modulation of differentiation. Recently it has been shown that ectopic expression of HH gene family members in vivo blocks chondrocyte maturation through activation of a parathyroid hormone related peptide (PTHrP) dependent negative regulatory loop in the perichondrium. However, the direct effect of HH on chondrocyte maturation has not been tested. Here, we studied the effect of retroviral overexpression of the chicken sonic hedgehog gene (Shh) on the growth and maturation of limb bud cells in micromass cultures. Shh is neither expressed nor required for the initiation of cellular condensation in normal micromass cultures. With Shh over-expression, micromass cultures developed novel tightly whorled nodules in addition to the normal Alcian Blue positive cartilage nodules. We characterized the new nodules and showed that they are strongly positive for alkaline phosphatase, enriched in type X collagen and weakly positive for Alcian Blue staining. Shh overexpression also increased cell proliferation, but this cannot account for the formation of the new nodules. This current study shows that misexpression of Shh in in vitro chondrogenic cultures promotes characteristics of hypertrophic chondrocytes. Thus HH has two complementary functions; a direct positive effect on chondrocyte hypertrophy in the absence of PTHrP pathway, and an indirect negative feedback loop through PTHrP to prevent other less differentiated chondrocytes from becoming hypertrophic. These two complementary actions of HH coordinate the progression of cartilage maturation.
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PMID:Dual action of sonic hedgehog on chondrocyte hypertrophy: retrovirus mediated ectopic sonic hedgehog expression in limb bud micromass culture induces novel cartilage nodules that are positive for alkaline phosphatase and type X collagen. 942 87

The NH2-terminal domain of sonic hedgehog (residue 25-198) was expressed in both yeast and animal cells. The yeast-derived NH2-terminal domain of sonic hedgehog was less active by far than the animal cell-derived counterpart. The yeast-derived NH2-terminal domain of sonic hedgehog lacked 10 amino acids from the NH2-terminus. This cleavage of the yeast-derived NH2-terminal domain of sonic hedgehog might due to Kex 2. In contrast, a mutant yeast-derived NH2-terminal domain of sonic hedgehog (Lys-33 to Thr) retained its NH2-terminus and its activity was comparable to that of the animal cell-derived NH2-terminal domain of sonic hedgehog. The NH2-terminal deleted NH2-terminal domain of sonic hedgehog completely lost its activity, nevertheless it inhibited the alkaline phosphatase activity induced by the animal cell-derived NH2-terminal domain of sonic hedgehog in a dose-dependent manner. These data suggest that the NH2-terminal deleted NH2-terminal domain of sonic hedgehog retains a receptor-binding ability and that the NH2-terminal peptide of the NH2-terminal domain of sonic hedgehog is necessary for its signal transduction.
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PMID:The NH2-terminal region of the active domain of sonic hedgehog is necessary for its signal transduction. 1021 71

Several lines of evidence implicate zinc finger proteins of the Gli family in the final steps of Hedgehog signaling in normal development and disease. C-terminally truncated mutant GLI3 proteins are also associated with human syndromes, but it is not clear whether these C-terminally truncated Gli proteins fulfil the same function as full-length ones. Here, structure-function analyses of Gli proteins have been performed using floor plate and neuronal induction assays in frog embryos, as well as induction of alkaline phosphatase (AP) in SHH-responsive mouse C3H10T1/2 (10T1/2) cells. These assays show that C-terminal sequences are required for positive inducing activity and cytoplasmic localization, whereas N-terminal sequences determine dominant negative function and nuclear localization. Analyses of nuclear targeted Gli1 and Gli2 proteins suggest that both activator and dominant negative proteins are modified forms. In embryos and COS cells, tagged Gli cDNAs yield C-terminally deleted forms similar to that of Ci. These results thus provide a molecular basis for the human Polydactyly type A and Pallister-Hall Syndrome phenotypes, derived from the deregulated production of C-terminally truncated GLI3 proteins. Analyses of full-length Gli function in 10T1/2 cells suggest that nuclear localization of activating forms is a regulated event and show that only Gli1 mimics SHH in inducing AP activity. Moreover, full-length Gli3 and all C-terminally truncated forms act antagonistically whereas Gli2 is inactive in this assay. In 10T1/2 cells, protein kinase A (PKA), a known inhibitor of Hh signaling, promotes Gli3 repressor formation and inhibits Gli1 function. Together, these findings suggest a context-dependent functional divergence of Gli protein function, in which a cell represses Gli3 and activates Gli1/2 prevents the formation of repressor Gli forms to respond to Shh. Interpretation of Hh signals by Gli proteins therefore appears to involve a fine balance of divergent functions within each and among different Gli proteins, the misregulation of which has profound biological consequences.
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PMID:Gli proteins encode context-dependent positive and negative functions: implications for development and disease. 1037 10

Hedgehog signaling is considered to play a crucial role in chondrogenesis by regulation through a network of cytokine actions, which is not fully understood. We examined the effect of hedgehog signaling on the expression of core-binding factor a1 (Cbfa1), a critical transcription factor for the development of bone and cartilage. Primary chondrocytes prepared from the costal cartilage of newborn mice were treated with N-terminal fragment of recombinant murine sonic hedgehog (rmShh-N). Northern blot analysis indicated that Cbfa1 mRNA expression levels in the chondrocyte cultures were elevated by the treatment with rmShh-N. rmShh-N treatment enhanced 1.8 kb Cbfa1 promoter activity in chondrocytes, suggesting the presence of transcriptional control. As Cbfa1-binding site(s) have been located in the promoter of the receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) gene, we also examined RANKL expression. rmShh-N treatment upregulated RANKL and RANK mRNA expression levels in chondrocytes. Interestingly, RANKL suppressed the hedgehog enhancement of alkaline phosphatase activity in chondrocytes, suggesting the presence of a link between these signaling molecules. We conclude that hedgehog signaling activates Cbfa1 gene expression through its promoter in chondrocytes, and also activates and interacts with RANKL to maintain cartilage development.
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PMID:Hedgehog signaling enhances core-binding factor a1 and receptor activator of nuclear factor-kappaB ligand (RANKL) gene expression in chondrocytes. 1277 22

We used both clonal osteoblast-like cells and primary calvarial osteoblastic cells to examine the role of Hedgehog in osteoblast biology. Primary osteoblasts and several clonal osteoblast-like cell lines express Indian hedgehog (Ihh), and genes encoding both components of its receptor, patched (Ptc) and smoothened (Smo). Moreover, Ihh is relatively increased in phenotypically mature clonal cells and it increases by fivefold in primary osteoblasts as they mature in culture. Recombinant N-terminal Sonic Hedgehog (rSHH-N) upregulates Ptc and Gli-1 in osteoblasts, classical transcriptional targets. Furthermore; in response to rSHH-N, immunoreactive parathyroid hormone-related peptide (iPTHrP) secretion is transiently increased in medium conditioned by primary osteoblasts. Changes in PTHrP expression mirror those of iPTHrP, except in late cultures, when mRNA levels remain relatively elevated in response to rSHH-N. Gli-1, but not Ptc, becomes resistant to treatment with rSHH-N over a time course paralleling that of PTHrP, suggesting that mechanisms regulated by Gli-1 affect PTHrP. Last, rSHH-N increases formation of mineralized bone nodules and it accelerates expression of alkaline phosphatase, alkaline phosphatase activity, and mineralization. Taken together, these data suggest a functional role for Hedgehog protein in osteoblast recruitment and differentiation, which includes stimulation of PTHrP expression and secretion.
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PMID:Hedgehog promotes primary osteoblast differentiation and increases PTHrP mRNA expression and iPTHrP secretion. 1281 Jan 68

The involvement of hedgehog signaling in the initiation of osteoblastic differentiation in the bone collar during endochondral bone formation has been well established. The stages at which hedgehog acts during osteoblast differentiation as well as its molecular mechanism of action are less well understood. To address these questions, we have made use of the preosteoblastic cell line KS483. First, a systematic survey of mRNA expression of osteoblastic differentiation showed expression of Ihh and signaling intermediates at all stages. Interestingly, expression of Ihh, Gli1 and Ptc1 peaked during the maturation phase. Addition of recombinant human sonic hedgehog (rShh) potently increased osteoblastic differentiation of KS483 cells dose-dependently as assayed by a modest increase in alkaline phosphatase (ALP) activity, a strong increase in matrix mineralization, and increased mRNA expression of established osteoblast marker genes. These effects were blocked by the hedgehog antagonist cyclopamine, which by itself was ineffective. Addition of rShh during early stages was sufficient, while addition to mature osteoblasts had no effect. Furthermore, hedgehog signaling could be completely blocked by the BMP antagonists, soluble truncated BMPR-IA and noggin. In contrast, the BMP-induced differentiation of KS483 cells could only be partly inhibited by high doses of cyclopamine. These data demonstrate that Hh-induced osteoblastic differentiation requires functional BMP signaling. In KS483 cells, Hh and BMP synergistically induced alkaline phosphatase activity only when suboptimal concentrations of BMP were used. This synergy did not occur at the level of immediate early BMP response, but at the level of Hh response as determined by transient transfection studies using either a BMP reporter or a Gli reporter construct. In addition, rShh inhibited adipogenesis of KS483 cells cultured under adipogenic culture conditions, suggesting that Hh is involved in directing differentiation of KS483 cells toward osteoblasts at the expense of adipogenesis. Using in situ hybridization, we demonstrated, for the first time, Ihh mRNA expression in vivo in osteoblasts and lining cells in the humerus of developing human skeleton. Our in vitro and in vivo data indicate a stimulatory role for osteoblast-expressed Ihh in bone formation in a positive feedback loop. It may recruit progenitor cells in the osteoblastic lineage at the expense of adipocytes and it may stimulate maturation of early osteoblasts.
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PMID:Hedgehog stimulates only osteoblastic differentiation of undifferentiated KS483 cells. 1467 49

During embryonic and cancer development, the Hedgehog family of proteins, including Sonic Hedgehog, play an important role by relieving the inhibition of Smo by Ptc, thus activating the Smo signaling cascade. Recently, a purine compound, purmorphamine, has been reported to target the Hedgehog signaling pathway by interacting with Smo. Interestingly, both Sonic Hedgehog and purmorphamine were found to promote the osteogenic differentiation of mouse chondroprogenitor cells. However, there is insufficient information as to how the activation of this seemingly unrelated signaling pathway, either by Sonic Hedgehog or purmorphamine, contributes to osteogenesis. Using alkaline phosphatase assays, we screened 125 purmorphamine derivatives from the Korea Chemical Bank for effects on the differentiation of preosteoblast C2C12 cells. Here, we report that two purine derivatives modulate ALP activity as well as the expression of genes whose expression is known or suggested to be involved in osteogenesis.
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PMID:Identification of osteogenic purmorphamine derivatives. 1869 57

In order to study the effects of photoperiod on fish bone, Atlantic salmon (Salmo salar L.) were exposed to two light regimes (natural and continuous light) from January until June. During the experimental period, several parameters related to the inorganic (minerals) and organic (osteoid) phases were measured. Changes in the organic phase were related to mechanical strength (yield-load) and the expression of the genes sonic hedgehog (shh) and collagen type I alpha 2 (col I). Co-variation between yield-load and the expression of both shh and col I were detected in both groups. It was also shown that fish on the continuous light regime had delayed activation of osteoid incorporation. Mineralization properties were measured with stiffness, mineral incorporation per day and expression of alkaline phosphatase (alp) and matrix Gla protein (mgp). Stiffness, mineral incorporation and gene expression followed the same trend in both light groups in late spring, whereas an increase in the expression of mgp and alp was detected in April, followed by significantly higher stiffness at last sampling in both light groups. These results indicate that constant light affects mineralization and delays osteoid incorporation in Atlantic salmon during the spring. However, in this experiment light treatment did not promote the development of vertebral deformities. Our results also suggest that shh can be used as a marker of osteoblast proliferation and col I a marker of osteoid incorporation, and that both alp and mgp expression could be associated with a rapid increase in mineralization in Atlantic salmon vertebrae.
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PMID:Continuous light affects mineralization and delays osteoid incorporation in vertebral bone of Atlantic salmon (Salmo salar L.). 1921 16

A number of previous studies documented the angiogenic potential of outgrowth endothelial cells in vitro and in vivo and provided evidence that therapeutic success could depend on coculture or coimplantation strategies. Thus, deeper insight into the molecular mechanisms underlying this pro-angiogenic effect of cocultures might provide new translational options for tissue engineering and regenerative medicine. One promising signaling pathway in bone repair involved in neoangiogenesis and bone formation is the sonic hedgehog (Shh) pathway. In this article, we focus on the effect of Shh on the formation of microvessel-like structures and osteoblastic differentiation in cocultures of primary osteoblasts and outgrowth endothelial cells. Already after 24 h of treatment, Shh leads to a massive increase in microvessel-like structures compared with untreated cocultures. Increased formation of angiogenic structures seems to correlate with the upregulation of vascular endothelial growth factor or angiopoietins (Ang-1 and Ang-2) studied at both the mRNA and protein levels. In addition, treatment with cyclopamine, an inhibitor of hedgehog signaling, blocked the formation of microvessel-like structures in the cocultures. However, exogenous Shh also resulted in the upregulation of several osteogenic differentiation markers in real-time polymerase chain reaction, as well as in an increased mineralization and alkaline phosphatase activity. The present data highlight the central role of the Shh pathway in bone regeneration and vascularization. Further, Shh might have the potential to improve both angiogenesis and osteogenesis in clinical applications in the future.
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PMID:Sonic hedgehog promotes angiogenesis and osteogenesis in a coculture system consisting of primary osteoblasts and outgrowth endothelial cells. 1988 47


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