Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M611741200 on January 2, 2007

J. Biol. Chem., Vol. 282, Issue 12, 8959-8968, March 23, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/12/8959    most recent
M611741200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dwyer, J. R.
Right arrow Articles by Parhami, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dwyer, J. R.
Right arrow Articles by Parhami, F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Oxysterols Are Novel Activators of the Hedgehog Signaling Pathway in Pluripotent Mesenchymal Cells*

Jennifer R. Dwyer{ddagger}, Navdar Sever§1, Marc Carlson, Stanley F. Nelson, Philip A. Beachy§2, and Farhad Parhami{ddagger}3

From the Departments of {ddagger}Medicine and Human Genetics, David Geffen School of Medicine, UCLA, Los Angeles, California 90095, and the §Department of Developmental Biology, Stanford University School of Medicine, Stanford, California 94305

Received for publication, December 21, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Pluripotent mesenchymal cells form a population of precursors to a variety of cell types, including osteoblasts and adipocytes. Aging tilts the balance in favor of adipocyte differentiation at the expense of osteoblast differentiation, resulting in reduced bone formation and osteopenic disorders, including osteoporosis, in humans and animals. Understanding the mechanisms involved in causing this apparent shift in differentiation and identifying factors that stimulate osteoblast formation while inhibiting adipogenesis are of great therapeutic interest. In this study we report that specific, naturally occurring oxysterols, previously shown to direct pluripotent mesenchymal cells toward an osteoblast lineage, exert their osteoinductive effects through activation of Hedgehog signaling pathway. This was demonstrated by 1) oxysterol-induced expression of the Hh target genes Gli-1 and Patched, 2) oxysterol-induced activation of a luciferase reporter driven by a multimerized Gli-responsive element, 3) inhibition of oxysterol effects by the hedgehog pathway inhibitor, cyclopamine, and 4) unresponsiveness of Smoothened-/- mouse embryonic fibroblasts to oxysterols. Using Patched-/- cells that possess high baseline Gli activity, we found that oxysterols did not dramatically shift the IC50 concentration of cyclopamine needed to inhibit Gli activity in these cells. Furthermore, binding studies showed that oxysterols did not compete with fluorescently labeled cyclopamine, BODIPY-cyclopamine, for direct binding to Smoothened. These findings demonstrate that oxysterols stimulate hedgehog pathway activity by indirectly activating the seven-transmembrane pathway component Smoothened. Osteoinductive oxysterols are, therefore, novel activators of the hedgehog pathway in pluripotent mesenchymal cells, and they may be important modulators of this critical signaling pathway that regulates numerous developmental and post-developmental processes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The Hedgehog (Hh)4 signaling pathway is indispensable for normal patterning of multicellular embryos and functions in postembryonic development and in adult tissue homeostasis, including regulation of stem cell physiology (1–3). Reduced Hh pathway activity can cause severe developmental defects in mice and humans, and aberrant increases in Hh pathway activity are associated with cancer (4, 5). Given these roles in various pathological conditions, small molecules that modulate Hh pathway activity represent potential therapeutic agents as well as pharmacological tools for probing the mechanisms of signal transduction.

Hh signaling begins with the release from producing cells of a doubly lipid-modified Hh protein that binds to the 12-transmembrane-spanning receptor Patched (Ptch) on Hh-responsive cells, relieving suppression of the 7-transmembrane protein, Smoothened (Smo). Smo in turn activates an intracellular signaling cascade that results in activation of one or more Gli transcription factors, which in turn mediate the transcription of the Hh target genes, including Gli-1 and Ptch. Mice lacking Sonic hedgehog (Shh) function fail to develop most craniofacial bones and exhibit marked defects in the skeletal derivatives of the sclerotome, including absence of a vertebral column and calcified ribs (6). Mice lacking function of the Indian hedgehog (Ihh) gene display defects in endochondral bone patterning and a deficit in osteoblast formation, resulting in stunted and disorganized limbs (7). These defects indicate a requirement for Hh signaling in the specification and differentiation of the axial and appendicular skeleton. Several studies have demonstrated the lineage-specific differentiation of pluripotent mesenchymal stem cells into osteoblasts upon Hh protein stimulation (8–10). Furthermore, the expression of molecules that stimulate osteoblast differentiation, including bone morphogenetic proteins-2, -4, and -7 (11, 12) and Wnt7b (13), is regulated by Hh signaling. Due to the decline in osteoprogenitor and osteoblast numbers and function observed in conditions such as senile and postmenopausal osteoporosis (14), identification of molecules with the potential to stimulate the differentiation of osteoprogenitors into mature, bone-forming osteoblasts is of great therapeutic interest (15, 16).

Oxysterols are products of cholesterol oxidation found in the circulation and in tissues. Oxysterols can be derived from the diet or from endogenous cellular biosynthesis, and they play important roles in physiological and pathological processes. We previously reported that specific oxysterols, namely 20(S)-hydroxycholesterol in combination with 22(S)- or 22(R)-hydroxycholesterol have potent osteoinductive properties when applied to marrow stromal cells (17, 18). This effect was evidenced from increased DNA binding activity of Runx2, the transcriptional regulator of osteoblastic gene expression, increased alkaline phosphatase (ALP) activity, important in mineralization of extracellular matrix and increased expression of osteocalcin (OCN), an osteoblast-specific gene. Significant increases in mineralized matrix, the ultimate measure of osteoblast function, were also observed with oxysterol treatment (17). Here we report that the osteoinductive effects of oxysterols are mediated by activation of the Hh signaling pathway. Furthermore, competition and binding studies strongly suggest that the effect of oxysterols on Hh pathway activity occurs upstream of Smo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Reagents—M2-10B4 cells, C3H10T1/2 cells, Smo-/- mouse embryonic fibroblasts (MEFs), and Ptch-/- MEFs were maintained as previously described (16, 17, 19, 20). Treatments were performed in differentiation medium containing 5% fetal bovine serum, 50 µg/ml ascorbate, and 3 mM beta-glycerophosphate. Oxysterols and phorbol 12-myristate 13-acetate (PMA) were obtained from Sigma-Aldrich, Co., cyclopamine and 3-keto-N-(aminoethylaminocaproyldihydrocinnamoyl) (KAAD)-cyclopamine were from EMD Biosciences, Inc. La Jolla, CA), recombinant mouse Shh N-terminal peptide and Shh-neutralizing antibody were from R&D Systems, Inc. (Minneapolis, MN), rottlerin and H-89 were from Calbiochem, and all antibodies for Western blotting were from Cell Signaling Technology (Danvers, MA). The plasmid pACMV-tetO and HEK293S-TetR cells were gifts from P. J. Reeves and H. G. Khorana (University of Essex, Colchester, UK). The polyclonal anti-Myc antibody was from Santa Cruz Biotechnology, and the enhanced chemiluminescence detection kit was from Amersham Biosciences. BODIPY-cyclopamine was from TRC (North York, Ontario, Canada), tetracycline was purchased from Sigma, and sodium butyrate was from J. T. Baker (Mallinchrodt Baker, Phillipsburg, NJ). Blasticidin and Geneticin were from Invitrogen.

Microarray—All samples were processed, scanned, and quality-checked on Affymetrix HG-U133A arrays (21). For analysis of gene expression measures, all Affymetrix data were normalized using model-based expression and the pair-matched-mismatched method from dChip (22). Subsequent to this, probe sets that showed at least a 2-fold change in expression, a minimum difference in expression of 100, and a two-sided t test p value of <0.01 between the two groups were selected for further analysis. Comparisons were made for all experimental versus all control and for experimental versus control comparisons at 8- and 48-h time points specifically. The lists generated in this way were then put through an EASE analysis (23) to test for enrichment of gene ontology terms. EASE analysis indicated an enrichment in terms for steroid metabolism in the 8-h comparison and for an enrichment of morphogenesis and developmental terms in the 48-h comparison.

Quantitative Real-time PCR—Q-RT-PCR was performed using reverse-transcribed RNA isolated from M2 cells using the phenol/chloroform method. PCR reactions were performed using iQ SYBR Green Supermix and an iCycler RT-PCR Detection System (Bio-Rad). Primer sequences for Gli-1, Gli-2, Gli-3, Shh, and Ihh were kindly provided by Dr. Fanxin Long (Washington University, St. Louis, MO). Ptch and Smo primer sequences are available upon request. Q-RT-PCR data were normalized to cyclophilin expression (24), and relative expression levels were calculated using the 2{Delta}{Delta}CT method (25).

Transient Transfection—Cells were plated into 24-well plates and transfected the next day with Gli-dependent firefly luciferase and Renilla luciferase vectors and where indicated, Smo or Ptch expression vectors. Total DNA per well did not exceed 500 ng, and FuGENE 6 transfection reagent (Roche Applied Science) was used at a ratio of 3:1 (reagent:DNA). Cells were treated for 48 h prior to assessing luciferase activity using the Dual Luciferase Reporter Assay System (Promega, Madison, WI) according to the manufacturer's instructions. Experiments were performed in triplicate, and error bars indicate one standard deviation.

Electrophoretic Mobility Shift Assay—The sequence of the OSE2 oligonucleotide was (5'-AGCTG CAATC ACCAA CCACA GCA-3') (26). Oligonucleotides were annealed to their complementary sequences by boiling and cooling. The probes were end-labeled with [{gamma}-32P]ATP using polynucleotide kinase and column-purified. Nuclear extracts were prepared using the modified Dignam protocol (27). Nuclear extracts (10 µg) were incubated in binding buffer (10 mM Tris, pH 7.5, 100 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 4% glycerol), 1 µg of poly-(dIdC), and 0.2 ng of labeled probe for 20 min at room temperature, and complexes were resolved on a cooled, 6% acrylamide 1x TBE (1 M Trizma base + 1 M boric acid + 20 mM EDTA) gel. Subsequently, gels were dried and exposed to film.

Alkaline Phosphatase Activity Assay, Northern Blotting, and Mineralization Assay—Colorimetric alkaline phosphatase activity assay on whole cell extracts was performed as previously described (17). Northern blotting for OCN and 18 S rRNA was performed as previously described (17). Gene expression was quantified using a Storm840 PhosphorImager and Image-QuaNT software (Amersham Biosciences).

Construction of the Tetracycline-regulated Smo Expression Plasmid, pACMV-tetO-Smo-Myc—The Smo-Myc gene was amplified from the plasmid pGE-Smo-Myc (20) using the primers 5'-AAAAT GAATT CAACA ACTCC GCCCC ATTGA C-3' and 5'-CCCGC GCGGC CGCCG ACTAC GACCT AATTC CTGC-3'. The resulting PCR product was digested with HindIII to isolate the Smo-Myc gene, end-repaired by using the DNA polymerase I Klenow fragment, and then digested with NotI. The Smo-Myc gene was purified by agarose gel electrophoresis and inserted into the plasmid pACMV-tetO, as previously described (28), to give the vector pACMV-tetO-Smo-Myc.


Figure 1
View larger version (27K):
[in this window]
[in a new window]

  		FIGURE 1.
Osteogenic oxysterols activate the Hedgehog pathway. Q-RT-PCR of mRNA from M2 cells treated with control vehicle (C), 5 µM SS or 200 ng/ml Shh were analyzed for induction of the Hh target genes Gli-1 (a) and Ptch (b). Data from a representative experiment are reported as the mean of quadruplicate determination ± S.D. (p < 0.005 for C versus SS and Shh at all time points for Gli-1 and Ptch). c, Hh pathway activation as measured by Gli-dependent luciferase reporter (Gli-Luc) activity in M2 cells. Cells were pre-treated for 2 h with 4 µM cyclopamine (Cyc) or Me2SO (dimethyl sulfoxide) vehicle, followed by 48 h of treatment with control vehicle (C), 5 µM SS, or 200 ng/ml Shh (p < 0.002 for C versus SS and Shh Gli-Luc, and for SS and Shh each with versus without Cyc). d, non-osteoinductive oxysterols, 7-{alpha}-hydroxycholesterol (7-alpha-HC) and 7-keto-hydroxycholesterol (7-keto-HC), each used at 5 µM, as well as the LXR agonist TO-901317 (TO) were assessed in parallel with 5 µM osteoinductive oxysterols SS and 200 ng/ml Shh for induction of Gli-luc reporter activity.

 
Construction of Stable HEK293S Cell Lines for Tetracycline-induced Smo Gene Expression—HEK293S-TetR cells were maintained and stably transfected with pACMV-tetO-Smo-Myc as described (28). Individual Geneticin-resistant colonies were expanded and screened for Smo-Myc expression by analyzing solubilized cell extracts by SDS-PAGE followed by immunodetection with anti-Myc polyclonal antibody. One cell line exhibiting tetracycline-inducible Smoothened expression was chosen and expanded for use in all subsequent experiments.

BODIPY-Cyclopamine Binding Assay—HEK293S stable cell lines containing the inducible Smoothened gene were grown to confluence in medium containing Geneticin (2 mg/ml) by using 6-well plates. The growth medium was then replaced with fresh medium containing tetracycline (1 µg/ml) and sodium butyrate (5 mM). After 2 days, fluorescence binding assays using BODIPY-cyclopamine were conducted as previously described (29).


Figure 2
View larger version (33K):
[in this window]
[in a new window]

  		FIGURE 2.
The Hedgehog pathway inhibitor, cyclopamine, inhibits oxysterol-induced osteoblastic differentiation. a, alkaline phosphatase activity assay in M2 cells pre-treated with various doses of cyclopamine (Cyc) or Me2SO (dimethyl sulfoxide) vehicle for 2 h followed by treatment for 3 days with the oxysterol combination, SS. Results from a representative experiment are reported as the mean of quadruplicate determinations ± S.D. and normalized to protein concentrations (p < 0.001 for C versus SS and SS versus SS plus Cyc at all concentrations). b, electrophoretic mobility shift assay analysis for Runx2 DNA binding activity in M2 cells treated for 4 days with control vehicle or 5 µM SS following pre-treatment with 4 µM Cyc or Me2SO (dimethyl sulfoxide) vehicle for 2 h. The shifted band (arrow) was previously characterized as Runx2 by supershift analysis and competition studies. c, analysis of osteocalcin (OCN) mRNA expression by Northern blotting. M2 cells were pre-treated with 4 µM Cyc for 2 h followed by treatment with control vehicle (C)or5 µM SS for 8 days. Blots were quantitated by phosphorimaging and OCN expression was normalized to 18 S rRNA levels. d, 45Ca incorporation assay was used to measure mineralization in M2 cells pre-treated with 4 µM Cyc or Me2SO (dimethyl sulfoxide) vehicle for 2 h, and then treated with 5 µM SS for 14 days. Data from a representative experiment are reported as the mean of quadruplicate determinations ± S.D. and normalized to protein concentrations (p < 0.001 for C versus SS and SS versus SS plus Cyc at 0.5 µM and above).

 
Western Blotting—After treatments, cells were lysed in lysis buffer, protein concentrations were determined using the Bio-Rad protein assay, and SDS-PAGE was performed as previously described (17), probing for native and phosphorylated proteins.

Statistical Analyses—Computer-assisted statistical analyses were performed using the StatView 4.5 program. All p values were calculated using analysis of variance and Fisher's projected least significant difference significance test. A value of p < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Hedgehog Pathway Activation by Osteogenic Oxysterols—To elucidate the molecular mechanisms involved in the osteoinductive effects of oxysterols, we performed a microarray-based gene expression analysis using Affymetrix mouse 430A gene chips comparing mRNA expression in the pluripotent mouse marrow stromal cell line M2-10B4 (M2) following treatment with control vehicle or an optimum dose of the oxysterol combination, 20(S)- and 22(S)-hydroxycholesterol (SS) (5 µM, 1:1) for 8 and 48 h. Relative to vehicle-treated controls, oxysterol treatment induced expression of the Hh target genes Gli (GLI-Kruppel family member GLI, NM_010296 [GenBank] , 3.3-fold induction at 8 h, p = 0.0008, and 14-fold induction at 48 h, p = 0.0002) and Ptch (patched homolog, NM_008957 [GenBank] , 38-fold induction at 48 h, p = 0.0001, with no apparent induction at 8 h) relative to vehicle-treated controls. Q-RT-PCR analysis confirmed these findings and demonstrated a robust increase in Gli-1 expression at 8, 24, and 48 h, and in Ptch expression at 24 and 48 h (Fig. 1, a and b). No significant changes were found in Gli-2 or Gli-3 gene expression at these time points (data not shown). Cells showed similar responses with a recombinant form of the mouse Shh N-terminal signaling domain (ShhN) (Fig. 1, a and b).


Figure 3
View larger version (23K):
[in this window]
[in a new window]

  		FIGURE 3.
Effect of Shh-neutralizing antibody on oxysterol- and Shh-induced ALP activity. M2 cells were treated with control vehicle (C), 5 µM SS, or 200 ng/ml Shh in the absence or presence of 5 or 10 µg/ml of Shh-neutralizing antibody (Ab). Cells were cultured for 3 days, and the extracts were analyzed for alkaline phosphatase activity. Results from a representative experiment are reported as the mean of quadruplicate determinations ± S.D. and normalized to protein concentrations (p < 0.005 for C versus SS and Shh; p < 0.001 for Shh versus Shh plus Ab at both concentrations; p = 0.8 for SS versus SS plus Ab at both concentrations).

 
To further examine Hh pathway activation by oxysterols, a reporter assay using a luciferase reporter driven by a multimerized Gli-responsive element (5'-GAACACCCA-3', Gli-luc) was used (15). M2 cells transfected with Gli-luc and treated with SS showed a 5-fold increase in luciferase activity over control vehicle-treated cells (Fig. 1c). Similar results were noted upon treatment of cells with ShhN (200 ng/ml), and induction by oxysterols or ShhN was inhibited by pre-treatment with the Hh pathway inhibitor, cyclopamine (29). Induction of Gli reporter activity was not observed for non-osteoinductive oxysterols, including 7-{alpha}-hydroxycholesterol and 7-ketocholesterol (Fig. 1d) (17), thus further supporting a role for Hh pathway activity in oxysterol-induced osteogenesis.

Role of Liver X Receptor in Hh Pathway Activation—As specific oxysterols, including 20(S)- and 22(R)-hydroxycholesterol, are known agonists of the nuclear hormone receptor liver X receptor (LXR) (30), and because LXR is expressed in M2 cells (17), we examined whether activation of LXR could lead to increased Hh signaling. Gli-luc reporter assay showed no activation in cells treated with 1 or 5 µM of the synthetic LXR agonist, TO-901317 (TO) (Fig. 1d). This is consistent with our previous finding that activation of LXR in M2 cells by similar concentrations of TO does not induce, but actually inhibits, osteoblastic differentiation (17). Such potentially adverse activation of LXR by osteoinductive oxysterols, such as 20(S)-hydroxycholesterol, emphasizes the importance of developing strategies that would limit its concentration if used therapeutically for osteopenic disorders. Combination oxysterol treatment using 20(S)-hydroxycholesterol with 22(S)hydroxycholesterol, which is not an LXR agonist and appears to enhance the osteoinductive effects of 20(S)-hydroxycholesterol (17), is one such strategy.

Hh Pathway Activation Mediates Oxysterol-induced Osteoblastic Differentiation—To determine the functional role of Hh signaling in oxysterol-induced osteoblastic differentiation, the effect of cyclopamine on oxysterol-induced markers of osteoblastic differentiation in M2 cells was evaluated. We found that the substantial induction in ALP activity produced by SS treatment was significantly inhibited by cyclopamine in a dose-dependent manner (Fig. 2a). Similarly, electrophoretic mobility shift assay analysis demonstrated that cyclopamine completely inhibited the stimulation of Runx2 DNA binding activity in oxysterol-treated cells (Fig. 2b). Furthermore, oxysterol-induced expression of OCN, a Runx2 target gene, and increased mineralization in cultures of M2 cells, were inhibited by cyclopamine (Fig. 2, c and d). Altogether, these findings demonstrate that the Hh signaling pathway is essential for the osteoinductive effects of oxysterols.


Figure 4
View larger version (26K):
[in this window]
[in a new window]

  		FIGURE 4.
Hedgehog pathway activation in mouse embryonic fibroblasts. a, C3H10T1/2 cells were treated with control vehicle or 5 µM SS with and without pre-treatment with cyclopamine (Cyc). Samples were analyzed for alkaline phosphatase activity after 2 days (p < 0.001 for control versus SS, and for SS versus SS plus Cyc). b, oxysterol-induced Hh pathway activation in C3H10T1/2 cells as measured by Gli-luc reporter assay. Cells were treated with control vehicle or 5 µM SS with or without pre-treatment with 4 µM Cyc (p < 0.001 for control versus SS, and for SS versus SS plus Cyc Gli-luc). c, Gli-dependent luciferase reporter assay in Smo-/- MEFs. Cells were transfected with or without Smo expression vector and assessed for responsiveness to control vehicle, 5µM SS, or conditioned medium from ShhN-overexpressing cells (ShhN-CM, p < 0.001 for control versus ShhN-CM and SS with Smo expression vector). d, alkaline phosphatase assay of Smo-/- MEFs treated with various concentrations of SS or with 50 ng/ml bone morphogenetic protein 7 for 2 days (p < 0.001 for control versus bone morphogenetic protein 7). e, Gli-dependent luciferase reporter assay in Ptch-/- MEFs. Cells were transfected with or without Ptch expression vector and analyzed for their response to control vehicle, 5 µM SS or conditioned medium from ShhN-overexpressing cells (ShhN-CM, p < 0.001 for control versus SS and Shh-CM with Ptch expression vector). f, Cyc titration assay in Ptch-/- MEFs in the presence or absence of oxysterols. Similar concentrations of cyclopamine are required to inhibit Gli-dependent luciferase expression in Ptch-/- MEFs in the absence or presence of 5µM SS, as demonstrated by the percentage of maximum Hh pathway activation after 48 h of treatment. Data from a representative experiment are reported as mean ± S.D. of triplicate samples. g, 200 nM KAAD-cyclopamine blocked the binding of BODIPY-cyclopamine to Smo-expressing HEK293S cells, but 5 µM of 20(S)-hydroxycholesterol or 22(S)-hydroxycholesterol, alone or in combination, were unable to reduce BODIPY-cyclopamine binding. Nonspecific binding as defined by cellular BODIPY-cyclopamine levels in the absence of Smo expression is indicated by the dashed line.

 
Mechanism of Oxysterol-induced Hh Pathway Activation— To elucidate the mechanism by which oxysterols cause Hh pathway activation, we first examined whether oxysterols induce the expression of endogenous Hh molecules by M2 cells. Q-RT-PCR analysis showed that oxysterol treatment (5 µM SS) of M2 cells for up to 48 h produced no change in the low levels of Ihh mRNA present in vehicle-treated control cells, and that Shh mRNA in M2 cells was undetectable with or without oxysterol treatment (data not shown). Furthermore, an Shh-neutralizing antibody did not inhibit oxysterol-induced ALP activity in M2 cells, whereas it completely inhibited ALP activity induced by exogenously added ShhN (Fig. 3). These results suggest that oxysterols do not affect endogenous Hh expression levels and must therefore cause Hh pathway activation via a different mechanism, perhaps by modulating other members of the Hh signaling network such as Smo and/or Ptch.

To examine this possibility, MEFs from Smo-/- and Ptch-/- null mice were used. To demonstrate that MEFs from mutant mouse embryos are an appropriate model system to further characterize the mechanism of oxysterol-induced Hh pathway activity, we first tested the effects of osteogenic sterols on wild-type C3H10T1/2 MEFs. Similar to the pluripotent marrow stromal cells, we found that C3H10T1/2 cells undergo osteoblastic differentiation in response to oxysterols, as assessed by the induction of ALP activity (Fig. 4a) and Runx2 DNA binding activity (18). Treatment with oxysterols also induced Gli-luc activity in C3H10T1/2 cells, and this activity was inhibited by cyclopamine pre-treatment (Fig. 4b). In contrast to wild-type MEFs, Smo-/- MEFs had very low Gli-luc activity and were unresponsive to treatment with oxysterols or with conditioned medium containing ShhN (ShhN-CM) (Fig. 4c). Responsiveness to SS and ShhN-CM was restored by transfection of a Smo expression vector, with no change in baseline reporter activity (Fig. 4c). Smo-/- MEFs also failed to undergo osteoblastic differentiation in response to oxysterols (Fig. 4d), although treatment with bone morphogenetic protein 7 did induce ALP activity in Smo-/- MEFs, thus bypassing the requirement for Hh pathway activity and confirming the inherent ability of these cells to differentiate along the osteoblastic lineage (Fig. 4d). Studies using Ptch-/- MEFs, in which baseline Hh pathway activity is high due to constitutive Smo activity, demonstrated that neither oxysterols nor ShhN-CM induced further pathway activation (Fig. 4e). Reintroduction of Ptch into Ptch-/- cells re-established Smo regulation, reduced baseline Hh pathway activity, and restored sensitivity to oxysterols and ShhN-CM in pathway activation (Fig. 4e). These results indicate that oxysterol induction of Hh pathway activity requires Smo, and that further activation by oxysterols does not occur when Smo is fully active due to loss of Ptch.

We next examined the possibility that oxysterols may stimulate Hh pathway activity by directly binding to and activating Smo, as previously demonstrated for pathway agonists Smo agonist (SAG) (15) and purmorphamine (16). Gli-luc reporter activity in Ptch-/- MEFs can be suppressed in a dose-dependent manner by treatment with the Smo antagonist cyclopamine, which acts by directly binding to and inhibiting Smo (29) (Fig. 4f). If oxysterols act by binding to and activating Smo, then a shift in the effective concentration of cyclopamine required for pathway inhibition would be expected. For example, the IC50 of cyclopamine action is shifted by several orders of magnitude upon treatment with Hh pathway-activating concentrations of the Smo agonists SAG and purmorphamine (15, 16). We noted, however, that oxysterols did not cause dramatic shifts in the concentrations of cyclopamine required to inhibit Gli-luc activity in Ptch-/- MEFs (Fig. 4f), suggesting that oxysterol action is not directly antagonistic to that of cyclopamine. Furthermore, we tested whether oxysterols can compete for binding of a fluorescent derivative of cyclopamine, BODIPY-cyclopamine, to cells expressing Smo. Following induction of Smo expression in HEK293S cells stably transfected with an inducible Smo expression construct, cells were co-treated with oxysterols or the potent cyclopamine derivative KAAD-cyclopamine in the presence of BODIPY-cyclopamine and subjected to fluorescence-activated cell sorting analysis. We found that binding of BODIPY-cyclopamine to HEK293S cells overexpressing Smo was not affected by SS, whereas KAAD-cyclopamine dramatically reduced BODIPY-cyclopamine binding (Fig. 4g).

Role of PKC and PKA in Oxysterol-induced Hh Pathway Activation—We previously reported that oxysterol-induced osteoblastic differentiation of cells is mediated via protein kinase C (PKC)- and protein kinase A (PKA)-dependent mechanisms (18). The role of these signaling pathways in regulating the different markers of osteoblastic differentiation appears to be both specific and overlapping (18). To begin elucidating the possible role of PKC and PKA in mediating oxysterol-induced Hh pathway activation, we examined the effect of PKC and PKA inhibitors on markers of Hh pathway activation. Pretreatment of M2 cells with the PKC{delta}-selective inhibitor, rottlerin, previously found to inhibit osteoblastic differentiation induced by oxysterols, dose-dependently inhibited oxysterol-induced Gli-1 and Ptch mRNA expression (Fig. 5, a and b). Similarly, oxysterol-induced Gli-1 and Ptch expression was inhibited in cells whose PKC stores were depleted following overnight pretreatment with 1 µM PMA (Fig. 5, c and d). We next examined whether oxysterols induced PKC activation by assessing the levels of phosphorylated MARCKS, a PKC substrate, by Western blotting. Whole cell lysates from M2 cells treated for 10 min, 30 min, 2 h, 8 h, 24 h, or 48 h with 5 µM SS did not show any increase in phosphorylated MARCKS levels compared with control untreated cells (data not shown), whereas a 30-min treatment with PMA clearly induced MARCKS phosphorylation.


Figure 5
View larger version (32K):
[in this window]
[in a new window]

  		FIGURE 5.
Effect of PKC inhibition on oxysterol-induced expression of hedgehog target genes. a and b, M2 cells were pretreated for 2 h with control vehicle or rottlerin (Rot) at the concentrations indicated (micromolar). Next, oxysterol combination SS or control vehicle (C) was added, and after 24 h of treatments RNA was isolated and analyzed by Q-RT-PCR for Gli-1 (a) and Ptch (b) expression. Data from a representative experiment are reported as the mean of triplicate determination ± S.D. (p < 0.001 for C versus SS and for SS versus SS plus Rot at all Rot concentrations for both Gli-1 and Ptch expression, except for SS versus SS plus Rot1 Gli1 expression where p < 0.01). Rot alone at all concentrations tested had no significant effect on gene expression (data not shown). c and d, M2 cells were pretreated overnight with 1 µM PMA or control vehicle followed by the addition of SS or control vehicle (C). After 24 h of treatments, Gli-1 (c) and Ptch (d) mRNA expression was analyzed by Q-RT-PCR. Data from a representative experiment are reported as the mean of triplicate determinations ± S.D. (p < 0.001 for C versus SS and for SS versus PMA plus SS for both Gli-1 and Ptch expression).

 
To examine the possible role of PKA in oxysterol-induced Hh pathway activation, the effect of PKA inhibitor, H-89, previously found to inhibit the induction of some, but not all, markers of osteoblastic differentiation, on oxysterol-induced Gli-1 and Ptch mRNA expression was assessed by Q-RT-PCR. Results showed that pretreatment of M2 cells with H-89 (5–15 µM) did not inhibit oxysterol-induced Gli-1 or Ptch expression after 24 h of treatment (data not shown). Furthermore, treatment of M2 cells for 24 h with SS (5 µM) together with the PKA pathway activator, forskolin (10 µM), completely inhibited oxysterol-induced Gli-1 and Ptch expression (data not shown). Finally, Western blotting of whole cell lysates from oxysterol-treated cells showed no significant induction of phosphorylated PKA or phosphorylated CREB levels compared with control untreated cells at similar time points described above for the examination of phosphorylated MARCKS levels (data not shown). In contrast, a 30-min treatment with forskolin (10 µM) significantly induced phosphorylated CREB levels.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The present study clearly demonstrates that specific oxysterols induce their osteoinductive effects through activation of the Hh signaling pathway in pluripotent mesenchymal cells. Our data also strongly suggest that, despite a requirement for Smo in oxysterol-mediated Hh pathway activation, oxysterols do not bind to or activate Smo directly. We cannot rule out the possibility that oxysterols may bind Smo at a site distinct from that bound by cyclopamine. However, the inability of oxysterols to antagonize cyclopamine binding or inhibition of Smo suggests that oxysterols are unlikely to directly influence the conformational shift thought to be critical for Smo activation (19). Our evidence instead supports a more indirect effect of oxysterols on Smo activity. One possibility for such an effect is that oxysterols might alter Smo subcellular localization in a manner that facilitates activation (31) but without preventing cyclopamine binding and antagonism. Alternatively, oxysterols might exert their effects at a distinct level of the pathway, for example, by inhibiting Ptch activity. Such a possibility is particularly attractive given the presence within Ptch of a region homologous to the sterol-sensing domains first identified in proteins such as 3-hydroxy-3-methylglutaryl-CoA reductase and sterol-regulatory element-binding protein-cleavage activating protein. The sterol-sensing domain within these proteins is involved in sensing and responding to cellular sterol levels; in the case of sterol-regulatory element-binding protein-cleavage activating protein, direct binding of cholesterol has been demonstrated (32). Thus, it is conceivable that direct binding to an oxysterol could modulate Ptch activity. Alternatively, oxysterols could exert their effects through interactions with other proteins or by modulating physical properties of membranes. Moreover, because Ptch is related to the resistance nodulation cell division family of bacterial pumps and to the Niemann-Pick C1 protein, involved in intracellular cholesterol transport, it has been suggested that it might modulate Smo activity through regulating the distribution of a secondary hydrophobic Smo-interacting endogenous small molecule (20). Such activity of Ptch might be subject to regulation by oxysterols, allowing for the transport and/or interaction of an activating small molecule with Smo in the presence of oxysterols. However indirect, these effects ultimately would have to impact the activity of an Hh pathway component at or upstream of the level of Smo.

Our data also provide evidence for the involvement of PKC signaling in oxysterol-induced hedgehog pathway activation. Cross-talk between the Hh pathway and several kinases has been reported, including PKC{delta} (33), mitogen-activated protein kinase (33), phosphatidylinositol 3-kinase (34), and PKA (35, 36). Results from the present study using PKC inhibitor, rottlerin, and PKC depletion through PMA pretreatment, are consistent with the role of PKC in mediating oxysterol-induced Hh pathway activation and expression of Hh target genes, Gli1 and Ptch. Interestingly, similar to what has been reported for Shh (33), PKC does not appear to be a downstream target of oxysterols, but rather basal PKC activity appears to be essential for oxysterol-induced expression of Hh target genes. Furthermore, our data are consistent with the negative role of PKA pathway activation in Hh signaling as indicated by the ability of forskolin to completely inhibit oxysterol effects on Gli1 and Ptch expression. However, it must be noted that, despite having an inhibitory role on Hh pathway activation, PKA does play a stimulatory role in mediating some of the osteogenic effects of oxysterols (18). The pro-osteogenic role of PKA is likely to be independent of Hh pathway activation and is likely to target downstream effectors that mediate terminal differentiation of cells into osteoblasts. Similar to PKC, PKA signaling does not appear to be a direct downstream target of oxysterols, and it appears that basal PKA activity is involved in mediating oxysterol effects on osteoblastic differentiation. Although it must be noted that, because our studies were carried out using whole cell lysates, we cannot rule out any potential activation of PKA or PKC by oxysterols in specific cellular compartments.

Corcoran and Scott (37) recently reported a modest (2- to 3-fold) activation of Hh pathway activity by certain oxysterols in the cell line PZp53MED, thought to lack Ptch activity and derived from a medulloblastoma that arose in a Ptch+/- mouse. In our studies we observed oxysterol induction of pathway activity by as much as 20- to 30-fold in wild-type cells, and no induction beyond the high constitutive levels present in Ptch-/- fibroblasts. The differences in our observations may result from inherent differences in baseline regulation of Hh pathway signaling in the two cell types and do not affect our observation that oxysterols fail to antagonize cyclopamine binding to or inhibition of Smo (Fig. 4, f and g) nor the resulting conclusion that the major effect of osteogenic oxysterols on Hh pathway activation is likely not through direct action on Smo.

Our study provides evidence that specific oxysterols activate the Hh signaling in pluripotent mesenchymal cells as demonstrated by increases in Gli-1 and Ptch mRNA expression and Gli-luc reporter activity. Given that the identified oxysterols capable of activating Hh signaling are naturally occurring molecules (38, 39) and that pathway activity is affected in multiple cell types, the present findings have numerous implications in physiological and pathological processes. For example, the role of Hh signaling in skeletal development and maintenance may be regulated at least in part through the action of oxysterols derived from endogenous cellular synthesis or dietary means. We previously reported that non-toxic doses of a cholesterol biosynthetic pathway inhibitor, mevastatin, blocked osteoblastic differentiation of progenitor cells (40), and that sterol depletion by genetic or pharmacological means blocks the cellular response to Hh protein stimulation (41). These findings suggest the possibility that one or more products of the cholesterol biosynthetic pathway may be capable of modulating Hh pathway activity during embryonic and postembryonic bone formation, as well as in other settings. The present findings further support this possibility by demonstrating that endogenous oxysterols can induce Hh pathway activity and therefore may play roles in osteogenesis and in other developmental processes that are regulated by Hh signaling. The role of Hh pathway activity in pathological processes such as carcinogenesis and metastasis further suggests the possibility that specific oxysterols may play an important role in tumorigenesis. Oxysterols and factors involved in their synthesis and metabolism thus may be relevant not only to the regulation of cholesterol biosynthesis (42) but also to other physiological and pathological processes.


   FOOTNOTES
 
* This work was supported in part by NIAMS, National Institutes of Health (NIH) Grant RO1AR050426 and NIH Grant HL30568. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 A Philip O'Bryan Montgomery, Jr., M.D. fellow of the Damon Runyon Cancer Research Foundation. Back

2 An investigator of the Howard Hughes Medical Institute. Back

3 To whom correspondence should be addressed: David Geffen School of Medicine at UCLA, Center for the Health Sciences, BH-307, 10833 Le Conte Ave., Los Angeles, CA 90095. Tel.: 310-825-5729; Fax: 310-206-9133; E-mail: fparhami{at}mednet.ucla.edu.

4 The abbreviations used are: Hh, Hedgehog; Ptch, Patched; Smo, Smoothened; Shh, Sonic hedgehog; Ihh, Indian hedgehog; ALP, alkaline phosphatase; OCN, osteocalcin; M2, M2-10B4 cells; MEF, mouse embryonic fibroblast; ShhN, Shh N-terminal peptide; Q-RT-PCR, quantitative real-time PCR; LXR, liver X receptor; TO, TO-901307; PKA, protein kinase A; PKC, protein kinase C; MARCKS, myristoylated alanine-rich C kinase substrate; SS, 20(S)- and 22(S)-hydroxycholesterol; ShhN-CM, conditioned medium containing ShhN. Back


   ACKNOWLEDGMENTS
 
We are grateful to members of the Parhami laboratory, Dr. Woo-Kyun Kim, Christopher M. Amantea, and Vicente Meliton, for helpful discussions and technical support, and to Drs. Theodore J. Hahn and Sotirios Tetradis for critical review of the manuscript and insightful discussions.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Zhang, Y., and Kalderon, D. (2001) Nature 410, 599-604[CrossRef][Medline] [Order article via Infotrieve]
  2. Lai, K., Kaspar, B. K., Gage, F. H., and Schaffer, D. V. (2003) Nat. Neurosci. 6, 21-27[CrossRef][Medline] [Order article via Infotrieve]
  3. Machold, R., Hayashi, S., Rutlin, M., Muzumdar, M. D., Nery, S., Corbin, J. G., Gritli-Linde, A., Dellovade, T., Porter, J. A., Rubin, L. L., Dudek, H., McMahon, A. P., and Fishell, G. (2003) Neuron 39, 937-950[CrossRef][Medline] [Order article via Infotrieve]
  4. Mullor, J. L., Sanchez, P., and Altaba, A. R. (2002) Trends Cell Biol. 12, 562-569[CrossRef][Medline] [Order article via Infotrieve]
  5. Lum, L., and Beachy, P. A. (2004) Science 304, 1755-1759[Abstract/Free Full Text]
  6. Chiang, C., Litingtung, Y., Lee, E., Young, K. E., Corden, J. L., Westphal, H., and Beachy, P. A. (1996) Nature 383, 407-413[CrossRef][Medline] [Order article via Infotrieve]
  7. St-Jacques, B., Hammerschmidt, M., and McMahon, A. P. (1999) Genes Dev. 13, 2072-2086[Abstract/Free Full Text]
  8. Nakamura, T., Aikawa, T., Iwamoto-Enomoto, M., Iwamoto, M., Higuchi, Y., Pacifici, M., Kinto, N., Yamaguchi, A., Noji, S., Kurisu, K., and Matsuya, T. (1997) Biochem. Biophys. Res. Commun. 237, 465-469[CrossRef][Medline] [Order article via Infotrieve]
  9. Spinella-Jaegle, S., Rawadi, G., Kawai, S., Gallea, S., Faucheu, C., Mollat, P., Courtois, B., Bergaud, B., Ramez, V., Blanchet, A. M., Adelmant, G., Baron, R., and Roman-Roman, S. (2001) J. Cell Sci. 114, 2085-2094[Abstract/Free Full Text]
  10. van der Horst, G., Farih-Sips, H., Lowik, C. W. G. M., and Karperien, M. (2003) Bone 33, 899-910[Medline] [Order article via Infotrieve]
  11. Krishnan, V., Ma, Y. L., Moseley, J. M., Geiser, A. G., Friant, S., and Frolik, C. A. (2001) Endocrinology 142, 940-947[Abstract/Free Full Text]
  12. Kawai, S., and Sugiura, T. (2001) Bone 28, 54-61[Medline] [Order article via Infotrieve]
  13. Hu, H., Hilton, M. J., Tu, X., Yu, K., Ornitz, D. M., and Long, F. (2005) Development 132, 49-60[Abstract/Free Full Text]
  14. Chan, G. K., and Duque, G. (2002) Gerontology 48, 62-71[CrossRef][Medline] [Order article via Infotrieve]
  15. Chen, J. K., Taipale, J., Young, K. E., Maiti, T., and Beachy, P. A. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 14071-14076[Abstract/Free Full Text]
  16. Sinha, S., and Chen, J. (2006) Nat. Chem. Biol. 2, 29-30[CrossRef][Medline] [Order article via Infotrieve]
  17. Kha, H. T., Basseri, B., Shouhed, D., Richardson, J., Tetradis, S., Hahn, T. J., and Parhami, F. (2004) J. Bone Min. Res. 19, 830-840[CrossRef][Medline] [Order article via Infotrieve]
  18. Richardson, J. A., Amantea, C. M., Kianmahd, B., Tetradis, S., Lieberman, J. R., Hahn, T. J., and Parhami, F. (2006) J. Cell. Biochem., in press
  19. Taipale, J., Chen, J. K., Cooper, M. K., Wang, B., Mann, R. K., Milenkovic, L., Scott, M. P., and Beachy, P. A. (2000) Nature 406, 1005-1009[CrossRef][Medline] [Order article via Infotrieve]
  20. Taipale, J., Cooper, M. K., Maiti, T., and Beachy, P. A. (2002) Nature 418, 892-897[CrossRef][Medline] [Order article via Infotrieve]
  21. Freije, W. A., Castro-Vargas, F. E., Fang, Z., Horvath, S., Cloughesy, T., Liau, L. M., Mischel, P. S., and Nelson, S. F. (2004) Cancer Res. 64, 6503-6510[Abstract/Free Full Text]
  22. Li, C., and Wong, W. H. (2003) in The Analysis of Gene Expression Data: Methods and Software (Parmigiani, G., Garret, E. S., Irizarry, R. A., and Zeger, S. L., eds) pp. 120-141, Springer, New York
  23. Hosack, D. A., Dennis, G., Jr., Sherman, B. T., Lane, H. C., and Lempicki, R. A. (2003) Genome Biol. 4, R70[CrossRef][Medline] [Order article via Infotrieve]
  24. Zhang, Y., Kast-Woelbern, H. R., and Edwards, P. A. (2003) J. Biol. Chem. 278, 104-110[Abstract/Free Full Text]
  25. Livak, K. J., and Schmittgen, T. D. (2001) Methods 25, 402-408[CrossRef][Medline] [Order article via Infotrieve]
  26. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A., and Karsenty, G. (1997) Cell 89, 747-754[CrossRef][Medline] [Order article via Infotrieve]
  27. Osborn, L., Kunkel, S., and Nabel, G. J. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 2336-2340[Abstract/Free Full Text]
  28. Reeves, P. J., Kim, J.-M., and Khorana, H. G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 13413-13418[Abstract/Free Full Text]
  29. Chen, J. K., Taipale, J., Cooper, M. K., and Beachy, P. A. (2002) Genes Dev. 16, 2743-2748[Abstract/Free Full Text]
  30. Edwards, P. A., Kast, H. R., and Anisfeld, A. M. (2002) J. Lipid Res. 43, 2-12[Abstract/Free Full Text]
  31. Denef, N., Neubuser, D., Perez, L., and Cohen, S. M. (2000) Cell 102, 521-531[CrossRef][Medline] [Order article via Infotrieve]
  32. Goldstein, J. L., DeBose-Boyd, R. L., and Brown, M. S. (2006) Cell 124, 35-46[CrossRef][Medline] [Order article via Infotrieve]
  33. Riobo, N. A., Haines, G. M., and Emerson, C. P. (2006) Cancer Res. 66, 839-845[Abstract/Free Full Text]
  34. Riobo, N. A., Lu, K., Ai, X., Haines, G. M., and Emerson, C. P. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 4505-4510[Abstract/Free Full Text]
  35. Zhou, Q., Apionishev, S., and Kalderon, D. (2006) Genetics 173, 2049-2062[Abstract/Free Full Text]
  36. Svard, J., Henricson, K. H., Persson-Lek, M., Rozell, B., Lauth, M., Bergstrom, A., Ericson, J., Toftgard, R., and Teglund, S. (2006) Dev. Cell 10, 187-197[CrossRef][Medline] [Order article via Infotrieve]
  37. Corcoran, R. B., and Scott, M. P. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 8408-8413[Abstract/Free Full Text]
  38. Edwards, P. A., and Ericsson, J. (1999) Annu. Rev. Biochem. 68, 157-185[CrossRef][Medline] [Order article via Infotrieve]
  39. Lin, Y. Y., Welch, M., and Lieberman, S. (2003) J. Steroid Biochem. Mol. Biol. 85, 57-61[CrossRef][Medline] [Order article via Infotrieve]
  40. Parhami, F., Mody, N., Gharavi, N., Ballard, A. J., Tintut, Y., and Demer, L. L. (2002) J. Bone Min. Res. 17, 1997-2003[CrossRef][Medline] [Order article via Infotrieve]
  41. Cooper, M. K., Wassif, C. A., Krakowiak, P. A., Taipale, J., Gong, R., Kelley, R. I., Porter, F. D., and Beachy, P. A. (2003) Nat. Genet. 33, 508-513[CrossRef][Medline] [Order article via Infotrieve]
  42. Schroepfer, G. J. (2000) Physiol. Rev. 80, 361-554[Abstract/Free Full Text]

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
K. W. Park, H. Waki, W.-K. Kim, B. S. J. Davies, S. G. Young, F. Parhami, and P. Tontonoz
The Small Molecule Phenamil Induces Osteoblast Differentiation and Mineralization
Mol. Cell. Biol., July 15, 2009; 29(14): 3905 - 3914.
[Abstract] [Full Text] [PDF]

Home page
 Stem CellsHome page
M. Plaisant, C. Fontaine, W. Cousin, N. Rochet, C. Dani, and P. Peraldi
Activation of Hedgehog Signaling Inhibits Osteoblast Differentiation of Human Mesenchymal Stem Cells
Stem Cells, March 1, 2009; 27(3): 703 - 713.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. Kawamura, K. Hervold, F.-A. Ramirez-Weber, and T. B. Kornberg
Two Patched Protein Subtypes and a Conserved Domain of Group I Proteins That Regulates Turnover
J. Biol. Chem., November 7, 2008; 283(45): 30964 - 30969.
[Abstract] [Full Text] [PDF]

Home page
 J EndocrinolHome page
P. J King, L. Guasti, and E. Laufer
Hedgehog signalling in endocrine development and disease
J. Endocrinol., September 1, 2008; 198(3): 439 - 450.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
E. Dessaud, A. P. McMahon, and J. Briscoe
Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network
Development, August 1, 2008; 135(15): 2489 - 2503.
[Abstract] [Full Text] [PDF]

Home page
 IBMS BoneKEyHome page
D. A. Towler
Vascular Calcification: A Perspective On An Imminent Disease Epidemic
IBMS BoneKEy, February 1, 2008; 5(2): 41 - 58.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
G. W. Calloni, C. Glavieux-Pardanaud, N. M. Le Douarin, and E. Dupin
Sonic Hedgehog promotes the development of multipotent neural crest progenitors endowed with both mesenchymal and neural potentials
PNAS, December 11, 2007; 104(50): 19879 - 19884.
[Abstract] [Full Text] [PDF]

Home page
 Arterioscler. Thromb. Vasc. Bio.Home page
Z. Al-Aly, J.-S. Shao, C.-F. Lai, E. Huang, J. Cai, A. Behrmann, S.-L. Cheng, and D. A. Towler
Aortic Msx2-Wnt Calcification Cascade Is Regulated by TNF-{alpha} Dependent Signals in Diabetic Ldlr / Mice
Arterioscler. Thromb. Vasc. Biol., December 1, 2007; 27(12): 2589 - 2596.
[Abstract] [Full Text] [PDF]

Home page
 DevelopmentHome page
T. E. Willnow, A. Hammes, and S. Eaton
Lipoproteins and their receptors in embryonic development: more than cholesterol clearance
Development, September 15, 2007; 134(18): 3239 - 3249.
[Abstract] [Full Text] [PDF]

Home page
 ScienceHome page
R. Rohatgi, L. Milenkovic, and M. P. Scott
Patched1 Regulates Hedgehog Signaling at the Primary Cilium
Science, July 20, 2007; 317(5836): 372 - 376.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
282/12/8959    most recent
M611741200v1
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dwyer, J. R.
Right arrow Articles by Parhami, F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dwyer, J. R.
Right arrow Articles by Parhami, F.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 2007 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement