Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the ... more Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumba...
Estrogen is a crucial hormone for osteoclast inhibition and for preventing osteoporosis. However,... more Estrogen is a crucial hormone for osteoclast inhibition and for preventing osteoporosis. However, the hormone's role in osteoblast growth and differentiation remains unclear. The complexity of estrogen's role in guiding osteoblast behavior arises partly from crosstalk with other signaling pathways, including Wnt signaling. In this study, we show that the Wnt agonist, LiCl, induced Fhl1 gene expression, which substantially enhanced osteoblast differentiation. Staining with alizarin red revealed that MC3T3-E1 mineralization was enhanced by overexpression of Fhl1. In addition, Fhl1 promoted the expression of the osteogenic markers, Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN), whereas MC3T3-E1 cells with gene knockdown of Fhl1 exhibited limited mineralization and expression of Runx2, OCN, and OPN. We further demonstrate evidences from quantitative reverse transcription real-time polymerase chain reaction and reporter assay that Fhl1 expr...
Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) loc... more Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) locus by gene targeting in embryonic stem (ES) cells to create a conditional gene expression system in mice. In the targeted alleles, expression of these cDNAs should be blocked by a neomycin resistance selection cassette that is flanked by loxP sites. Transgene expression should be activated after the blocking cassette is deleted by Cre recombinase. ^ To test this conditional expression system, I have bred R26-stop- Sry and R26-stop-Wnt4 heterozygotes with a MisRII-Cre mouse line that expresses Cre in the gonads of both sexes. Analysis of these two types of bigenic heterozygotes indicated that their gonads developed normally like those of wild types. However, one XX R26-Sry/R26-Sry; MisR2-Cre/+ showed epididymis-like structures resembling those of males. In contrast, only normal phenotypes were observed in XY R26-Wnt4/R26-Wnt4; MisR2-Cre /+ mice. To interpret these results, I have tested for Cre recombinase activity by Southern blot and transcription of the Sry and Wnt4 transgenes by RT-PCR. Results showed that bigenic mutants had insufficient activation of the transgenes in their gonads at E12.5 and E13.5. Therefore, the failure to observe mutant phenotypes may have resulted from low activity of MisR2-Cre recombination at the appropriate time. ^ Col2a1-Cre transgenic mice express Cre in differentiating chondrocytes. R26-Wnt4; Col2a1-Cre bigenic heterozygous mice were found to exhibit a dramatic alteration in growth presumably caused by Wnt4 overexpression during chondrogenesis. R26-Wnt4; Col2a1-Cre mice exhibited dwarfism beginning approximately 10 days after birth. In addition, they also had craniofacial abnormalities, and had delayed ossification of the lumbar vertebrate and pelvic bones. Histological analysis of the growth plates of R26-Wnt4; Col2a1-Cre mice revealed less structural organization and a delay in onset of the primary and secondary ossification centers. Molecular studies confirmed that overexpression of Wnt4 causes decreased proliferation and early maturation of chondrocytes. In addition, R26-Wnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF), suggesting that defects in vascularization may contribute to the dwarf phenotype. Finally, 9-month-old R26-Wnt4; Col2a1-Cre mice had significantly more fat cells in the marrow cavities of their metaphysis long bones, implying that long-term overexpression of Wnt4may cause bone marrow pathologies. In conclusion, Wnt4 was activated by Col2a1-Cre recombinase and was overexpressed in the growth plate, resulting in aberrant proliferation and differentiation of chondrocytes, and ultimately leads to dwarfism in mice. ^
Http Dx Doi Org 10 1080 19768354 2013 856341, Dec 24, 2013
Wnt signaling is well known playing dual roles during chondrogenesis. The overexpression of Fhl1 ... more Wnt signaling is well known playing dual roles during chondrogenesis. The overexpression of Fhl1 (Four-and-a-half LIM domain 1) induces myotube formation as a downstream event of Wnt signaling. Because Fhl1 is widely expressed in other mesenchyme-derived tissues, including chondrocytes, we investigated the role of Fhl1 in chondrogenesis and its relationship with Wnt signaling. We found that the expression of Fhl1 can be enhanced by β-catenin and LiCl (Lithium chloride) in chondrogenic ATDC5 cells. Overexpression of Fhl1 as well as canonical Wnt signaling inhibits chondrogenesis of ATDC5 cells. Moreover, shRNA-mediated knockdown of Fhl1 expression also inhibited ATDC5 cell differentiation, and this result is resembled to the mutant phenotype caused by deletion of β-catenin as was described previously. Because the endogenous Fhl1 expression remains constant during the middle and late phases of chondrogenesis, we propose that proper Fhl1 expression is necessary for the chondrogenic differentiation of ATDC5 cells and altered Fhl1 expression serves as an aberrant Wnt signal that impedes chondrogenesis.
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1994
The transcriptional response to growth factors and other mitogenic signals is mediated by the ser... more The transcriptional response to growth factors and other mitogenic signals is mediated by the serum response elements (SREs) located in the promoters of many immediate early genes, including the c-fos and beta-actin genes. We investigated SRE-regulated transcription in cell cycle-synchronized nuclei and found that a SRE-regulated reporter gene was transcribed actively during G1 and, surprisingly, during G2-M as well. One possible mechanism involved in the latter event is microtubule reorganization. Microtubule disassembly is mimicked by microtubule-disrupting drugs, and we found that these drugs, including colchicine, nocodazole, and vinblastine, could activate SRE-dependent reporter genes, as well as the c-fos protooncogene, in asynchronously growing cells. Taken together, our results suggested a possible relationship between cytoplasmic microtubule dynamics and cell cycle gene expression. Although the detailed molecular mechanisms of drug action are not known, protein phosphorylat...
Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has... more Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has been extensively investigated. Monocytes and neutrophils are derived from a common myeloid progenitor; therefore, ELA2 mutations can also influence monocyte development. These effects have not been well described. In this study, we used the human monocytic THP-1, to carry the human wild-type and G185R mutant ELA2 gene. Growth, death, differentiation and BiP expression were evaluated in the two stable cell lines and in the wild-type THP-1 cells. Exogenous wild-type ELA2 markedly increased THP-1 differentiation, whereas G185R ELA2 was incompetent to promote THP-1 differentiation in response to all-trans retinoic acid (ATRA). Indeed, during differentiation induced by ATRA, G185R cell line showed significant cell death. Also, up-regulated BiP expression accompanied cell death in the G185R cells, suggesting that the overexpression of G185R elastase increases apoptosis through an unfolded protein response. The G185R cells treated with lithium chloride (LiCl; a Wnt signalling activator) displayed higher BiP expression but similar cell viability compared with THP1 and HNEwt/THP1 cells treated with LiCl. This suggested that Wnt signalling might increase cellular tolerance to endoplasmic reticulum stress, enabling mutant monocyte survival.
Previous studies have shown that Wnt signaling is involved in postnatal mammalian myogenesis; how... more Previous studies have shown that Wnt signaling is involved in postnatal mammalian myogenesis; however, the downstream mechanism of Wnt signaling is not fully understood. This study reports that the murine four-and-ahalf LIM domain 1 (Fhl1) could be stimulated by b-catenin or LiCl treatment to induce myogenesis. In contrast, knockdown of the Fhl1 gene expression in C2C12 cells led to reduced myotube formation. We also adopted reporter assays to demonstrate that either b-catenin or LiCl significantly activated the Fhl1 promoter, which contains four putative consensus TCF/LEF binding sites. Mutations of two of these sites caused a significant decrease in promoter activity by luciferase reporter assay. Thus, we suggest that Wnt signaling induces muscle cell differentiation, at least partly, through Fhl1 activation.
The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, ut... more The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding b-galactosidase (b-gal), into the AMHR-II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2-lacZ heterozygotes express b-gal activity in an Amhr2specific pattern. In the gonads, b-gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. b-gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, b-gal activity was restricted to one side of the ductal mesoepithelium, whereas in males b-gal expression encircled the duct. b-gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal b-gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2-expressing mesenchyme.
REVIEW ARTICLE. Neutrophil elastase in cyclic and severe congenital neutropenia. Marshall S. Horw... more REVIEW ARTICLE. Neutrophil elastase in cyclic and severe congenital neutropenia. Marshall S. Horwitz 1 , Zhijun Duan 1 , Brice Korkmaz 1 , Hu-Hui Lee 1 , Matthew E. Mealiffe 1 , and Stephen J. Salipante 2. 1 Division of Medical ...
Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the ... more Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumba...
Estrogen is a crucial hormone for osteoclast inhibition and for preventing osteoporosis. However,... more Estrogen is a crucial hormone for osteoclast inhibition and for preventing osteoporosis. However, the hormone's role in osteoblast growth and differentiation remains unclear. The complexity of estrogen's role in guiding osteoblast behavior arises partly from crosstalk with other signaling pathways, including Wnt signaling. In this study, we show that the Wnt agonist, LiCl, induced Fhl1 gene expression, which substantially enhanced osteoblast differentiation. Staining with alizarin red revealed that MC3T3-E1 mineralization was enhanced by overexpression of Fhl1. In addition, Fhl1 promoted the expression of the osteogenic markers, Runt-related transcription factor 2 (Runx2), osteocalcin (OCN), and osteopontin (OPN), whereas MC3T3-E1 cells with gene knockdown of Fhl1 exhibited limited mineralization and expression of Runx2, OCN, and OPN. We further demonstrate evidences from quantitative reverse transcription real-time polymerase chain reaction and reporter assay that Fhl1 expr...
Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) loc... more Sry and Wnt4 cDNAs were individually introduced into the ubiquitously-expressed Rosa26 ( R26) locus by gene targeting in embryonic stem (ES) cells to create a conditional gene expression system in mice. In the targeted alleles, expression of these cDNAs should be blocked by a neomycin resistance selection cassette that is flanked by loxP sites. Transgene expression should be activated after the blocking cassette is deleted by Cre recombinase. ^ To test this conditional expression system, I have bred R26-stop- Sry and R26-stop-Wnt4 heterozygotes with a MisRII-Cre mouse line that expresses Cre in the gonads of both sexes. Analysis of these two types of bigenic heterozygotes indicated that their gonads developed normally like those of wild types. However, one XX R26-Sry/R26-Sry; MisR2-Cre/+ showed epididymis-like structures resembling those of males. In contrast, only normal phenotypes were observed in XY R26-Wnt4/R26-Wnt4; MisR2-Cre /+ mice. To interpret these results, I have tested for Cre recombinase activity by Southern blot and transcription of the Sry and Wnt4 transgenes by RT-PCR. Results showed that bigenic mutants had insufficient activation of the transgenes in their gonads at E12.5 and E13.5. Therefore, the failure to observe mutant phenotypes may have resulted from low activity of MisR2-Cre recombination at the appropriate time. ^ Col2a1-Cre transgenic mice express Cre in differentiating chondrocytes. R26-Wnt4; Col2a1-Cre bigenic heterozygous mice were found to exhibit a dramatic alteration in growth presumably caused by Wnt4 overexpression during chondrogenesis. R26-Wnt4; Col2a1-Cre mice exhibited dwarfism beginning approximately 10 days after birth. In addition, they also had craniofacial abnormalities, and had delayed ossification of the lumbar vertebrate and pelvic bones. Histological analysis of the growth plates of R26-Wnt4; Col2a1-Cre mice revealed less structural organization and a delay in onset of the primary and secondary ossification centers. Molecular studies confirmed that overexpression of Wnt4 causes decreased proliferation and early maturation of chondrocytes. In addition, R26-Wnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF), suggesting that defects in vascularization may contribute to the dwarf phenotype. Finally, 9-month-old R26-Wnt4; Col2a1-Cre mice had significantly more fat cells in the marrow cavities of their metaphysis long bones, implying that long-term overexpression of Wnt4may cause bone marrow pathologies. In conclusion, Wnt4 was activated by Col2a1-Cre recombinase and was overexpressed in the growth plate, resulting in aberrant proliferation and differentiation of chondrocytes, and ultimately leads to dwarfism in mice. ^
Http Dx Doi Org 10 1080 19768354 2013 856341, Dec 24, 2013
Wnt signaling is well known playing dual roles during chondrogenesis. The overexpression of Fhl1 ... more Wnt signaling is well known playing dual roles during chondrogenesis. The overexpression of Fhl1 (Four-and-a-half LIM domain 1) induces myotube formation as a downstream event of Wnt signaling. Because Fhl1 is widely expressed in other mesenchyme-derived tissues, including chondrocytes, we investigated the role of Fhl1 in chondrogenesis and its relationship with Wnt signaling. We found that the expression of Fhl1 can be enhanced by β-catenin and LiCl (Lithium chloride) in chondrogenic ATDC5 cells. Overexpression of Fhl1 as well as canonical Wnt signaling inhibits chondrogenesis of ATDC5 cells. Moreover, shRNA-mediated knockdown of Fhl1 expression also inhibited ATDC5 cell differentiation, and this result is resembled to the mutant phenotype caused by deletion of β-catenin as was described previously. Because the endogenous Fhl1 expression remains constant during the middle and late phases of chondrogenesis, we propose that proper Fhl1 expression is necessary for the chondrogenic differentiation of ATDC5 cells and altered Fhl1 expression serves as an aberrant Wnt signal that impedes chondrogenesis.
Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research, 1994
The transcriptional response to growth factors and other mitogenic signals is mediated by the ser... more The transcriptional response to growth factors and other mitogenic signals is mediated by the serum response elements (SREs) located in the promoters of many immediate early genes, including the c-fos and beta-actin genes. We investigated SRE-regulated transcription in cell cycle-synchronized nuclei and found that a SRE-regulated reporter gene was transcribed actively during G1 and, surprisingly, during G2-M as well. One possible mechanism involved in the latter event is microtubule reorganization. Microtubule disassembly is mimicked by microtubule-disrupting drugs, and we found that these drugs, including colchicine, nocodazole, and vinblastine, could activate SRE-dependent reporter genes, as well as the c-fos protooncogene, in asynchronously growing cells. Taken together, our results suggested a possible relationship between cytoplasmic microtubule dynamics and cell cycle gene expression. Although the detailed molecular mechanisms of drug action are not known, protein phosphorylat...
Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has... more Deficiency in neutrophils (neutropenia) caused by mutations in neutrophil elastase (NE, ELA2) has been extensively investigated. Monocytes and neutrophils are derived from a common myeloid progenitor; therefore, ELA2 mutations can also influence monocyte development. These effects have not been well described. In this study, we used the human monocytic THP-1, to carry the human wild-type and G185R mutant ELA2 gene. Growth, death, differentiation and BiP expression were evaluated in the two stable cell lines and in the wild-type THP-1 cells. Exogenous wild-type ELA2 markedly increased THP-1 differentiation, whereas G185R ELA2 was incompetent to promote THP-1 differentiation in response to all-trans retinoic acid (ATRA). Indeed, during differentiation induced by ATRA, G185R cell line showed significant cell death. Also, up-regulated BiP expression accompanied cell death in the G185R cells, suggesting that the overexpression of G185R elastase increases apoptosis through an unfolded protein response. The G185R cells treated with lithium chloride (LiCl; a Wnt signalling activator) displayed higher BiP expression but similar cell viability compared with THP1 and HNEwt/THP1 cells treated with LiCl. This suggested that Wnt signalling might increase cellular tolerance to endoplasmic reticulum stress, enabling mutant monocyte survival.
Previous studies have shown that Wnt signaling is involved in postnatal mammalian myogenesis; how... more Previous studies have shown that Wnt signaling is involved in postnatal mammalian myogenesis; however, the downstream mechanism of Wnt signaling is not fully understood. This study reports that the murine four-and-ahalf LIM domain 1 (Fhl1) could be stimulated by b-catenin or LiCl treatment to induce myogenesis. In contrast, knockdown of the Fhl1 gene expression in C2C12 cells led to reduced myotube formation. We also adopted reporter assays to demonstrate that either b-catenin or LiCl significantly activated the Fhl1 promoter, which contains four putative consensus TCF/LEF binding sites. Mutations of two of these sites caused a significant decrease in promoter activity by luciferase reporter assay. Thus, we suggest that Wnt signaling induces muscle cell differentiation, at least partly, through Fhl1 activation.
The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, ut... more The Müllerian ducts give rise to the female reproductive tract, including the Fallopian tubes, uterus, cervix, and anterior vagina. In male embryos, the Müllerian ducts regress, preventing the formation of female organs. We introduced the bacterial lacZ gene, encoding b-galactosidase (b-gal), into the AMHR-II locus (Amhr2) by gene targeting in mouse embryonic stem (ES) cells to mark Müllerian duct differentiation and regression. We show that Amhr2-lacZ heterozygotes express b-gal activity in an Amhr2specific pattern. In the gonads, b-gal activity was detected in Sertoli cells of the testes from 2 weeks after birth, and fetal ovaries and granulosa cells of the adult ovary. b-gal activity was first detected in the rostral mesenchyme of the Müllerian ducts at 12.5 days post coitus (dpc) in both sexes but soon thereafter expression was found along the entire length of the Müllerian ducts with higher levels initially found in males. In females, b-gal activity was restricted to one side of the ductal mesoepithelium, whereas in males b-gal expression encircled the duct. b-gal activity was also detected in the coelomic epithelium at 13.5 and 14.5 dpc. In male embryos, mesenchymal b-gal activity permitted the visualization of the temporal and spatial pattern of Müllerian duct regression. This pattern was similar to that observed using a Müllerian duct mesoepithelium lacZ reporter, indicating a coordinated loss of Müllerian duct mesoepithelium and Amhr2-expressing mesenchyme.
REVIEW ARTICLE. Neutrophil elastase in cyclic and severe congenital neutropenia. Marshall S. Horw... more REVIEW ARTICLE. Neutrophil elastase in cyclic and severe congenital neutropenia. Marshall S. Horwitz 1 , Zhijun Duan 1 , Brice Korkmaz 1 , Hu-Hui Lee 1 , Matthew E. Mealiffe 1 , and Stephen J. Salipante 2. 1 Division of Medical ...
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