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XBP1S, a BMP2-inducible transcription factor, accelerates endochondral bone growth by activating GEP growth factor.

Guo FJ, Xiong Z, Han X, Liu C, Liu Y, Jiang R, Zhang P - J. Cell. Mol. Med. (2014)

Bottom Line: XBP1S stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo.Furthermore, XBP1S enhances GEP-stimulated chondrogenesis and endochondral bone formation.Collectively, these findings demonstrate that XBP1S, a BMP2-inducible transcription factor, positively regulates endochondral bone formation by activating GEP chondrogenic growth factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing, China.

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Related in: MedlinePlus

XBP1S activates the chondroinductive action of granulin-epithelin precursor (GEP). (A) Schematic structures of wild-type and mutant XBP1S proteins. The N-terminal domain (N-term), one DNA binding domain (type a domain) and one transactivating domain (type b domain) are indicated. RXSXL and RXTXK are the amino acid sequences in the DNA binding domain and the transactivating domain. In the XBP1Smt-a mutant, the RXSXL in the a domain was substituted by RXGXK, in the XBP1Smt-b mutant, the RXTXK in the b domain was substituted by RXCXE, and in the XBP1Smt-a/b double mutant, the RXSXL and RXTXK motifs in both a and b domains were replaced by RXGXK and RXCXE. (B) XBP1S improves GEP-induced Col X and MMP-13 expression, while XBP1S mutants failed to enhance the GEP-induced Col X and MMP-13 expression, assayed by real-time PCR. ATDC5 cells pre-treated with BMP2 for 5 days were cultured in the absence or presence of Ad-GEP (MOI20), Ad-XBP1S (MOI20)+Ad-GEP, Ad-XBP1Smt-a (MOI20)+Ad-GEP, Ad-XBP1Smt-b (MOI 20)+Ad-GEP, Ad-XBP1Smt-a/-b (MOI 20) + Ad-GEP as indicated for an additional 3 days, and Col X and MMP-13 expression was measured by real-time PCR. The units are arbitrary, and the normalized values were calibrated against controls, here given the value of 1. Asterisks indicate a significant difference from the control (P < 0.05). (C) XBP1S enhances GEP-stimulated endochondral ossification. (a and b) Safranin O-fast green staining of metatarsals. Metatarsals were explanted from 15-day-old mouse embryos and cultured in the absence (CTR) or presence of Ad-GEP (MOI 20) with or without Ad-XBP1S. After 5 days of culture, safranin O-fast green staining was observed by using low-power (a) or high-power (b) microphotography. (c) Alizarin red S and alcian blue staining of metatarsals. The explants were fixed and processed for alizarin red S and alcian blue staining; a representative photograph is presented. (d) Per cent increase in total and mineralization length of metatarsal bones. Metatarsals were cultured as described above, the total or mineralization length was determined and the per cent increase was calculated (per cent increase = [length at day 5 − length at day 0]/length at day 0). Asterisks indicate a significant difference from the control (P < 0.05).
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fig09: XBP1S activates the chondroinductive action of granulin-epithelin precursor (GEP). (A) Schematic structures of wild-type and mutant XBP1S proteins. The N-terminal domain (N-term), one DNA binding domain (type a domain) and one transactivating domain (type b domain) are indicated. RXSXL and RXTXK are the amino acid sequences in the DNA binding domain and the transactivating domain. In the XBP1Smt-a mutant, the RXSXL in the a domain was substituted by RXGXK, in the XBP1Smt-b mutant, the RXTXK in the b domain was substituted by RXCXE, and in the XBP1Smt-a/b double mutant, the RXSXL and RXTXK motifs in both a and b domains were replaced by RXGXK and RXCXE. (B) XBP1S improves GEP-induced Col X and MMP-13 expression, while XBP1S mutants failed to enhance the GEP-induced Col X and MMP-13 expression, assayed by real-time PCR. ATDC5 cells pre-treated with BMP2 for 5 days were cultured in the absence or presence of Ad-GEP (MOI20), Ad-XBP1S (MOI20)+Ad-GEP, Ad-XBP1Smt-a (MOI20)+Ad-GEP, Ad-XBP1Smt-b (MOI 20)+Ad-GEP, Ad-XBP1Smt-a/-b (MOI 20) + Ad-GEP as indicated for an additional 3 days, and Col X and MMP-13 expression was measured by real-time PCR. The units are arbitrary, and the normalized values were calibrated against controls, here given the value of 1. Asterisks indicate a significant difference from the control (P < 0.05). (C) XBP1S enhances GEP-stimulated endochondral ossification. (a and b) Safranin O-fast green staining of metatarsals. Metatarsals were explanted from 15-day-old mouse embryos and cultured in the absence (CTR) or presence of Ad-GEP (MOI 20) with or without Ad-XBP1S. After 5 days of culture, safranin O-fast green staining was observed by using low-power (a) or high-power (b) microphotography. (c) Alizarin red S and alcian blue staining of metatarsals. The explants were fixed and processed for alizarin red S and alcian blue staining; a representative photograph is presented. (d) Per cent increase in total and mineralization length of metatarsal bones. Metatarsals were cultured as described above, the total or mineralization length was determined and the per cent increase was calculated (per cent increase = [length at day 5 − length at day 0]/length at day 0). Asterisks indicate a significant difference from the control (P < 0.05).

Mentions: Foetal mouse metatarsals were dissected from foetal GEP mice (GEP−/−, 15-day-old embryos) and cultured in DMEM (Gibco, Carlsbad, CA, USA) containing 1% heat-inactivated foetal calf serum (Invitrogen) and 100 U penicillin-streptomycin per milliliter in the absence or presence of various stimuli for 5 days, as indicated in Figures4, 8 and 9.


XBP1S, a BMP2-inducible transcription factor, accelerates endochondral bone growth by activating GEP growth factor.

Guo FJ, Xiong Z, Han X, Liu C, Liu Y, Jiang R, Zhang P - J. Cell. Mol. Med. (2014)

XBP1S activates the chondroinductive action of granulin-epithelin precursor (GEP). (A) Schematic structures of wild-type and mutant XBP1S proteins. The N-terminal domain (N-term), one DNA binding domain (type a domain) and one transactivating domain (type b domain) are indicated. RXSXL and RXTXK are the amino acid sequences in the DNA binding domain and the transactivating domain. In the XBP1Smt-a mutant, the RXSXL in the a domain was substituted by RXGXK, in the XBP1Smt-b mutant, the RXTXK in the b domain was substituted by RXCXE, and in the XBP1Smt-a/b double mutant, the RXSXL and RXTXK motifs in both a and b domains were replaced by RXGXK and RXCXE. (B) XBP1S improves GEP-induced Col X and MMP-13 expression, while XBP1S mutants failed to enhance the GEP-induced Col X and MMP-13 expression, assayed by real-time PCR. ATDC5 cells pre-treated with BMP2 for 5 days were cultured in the absence or presence of Ad-GEP (MOI20), Ad-XBP1S (MOI20)+Ad-GEP, Ad-XBP1Smt-a (MOI20)+Ad-GEP, Ad-XBP1Smt-b (MOI 20)+Ad-GEP, Ad-XBP1Smt-a/-b (MOI 20) + Ad-GEP as indicated for an additional 3 days, and Col X and MMP-13 expression was measured by real-time PCR. The units are arbitrary, and the normalized values were calibrated against controls, here given the value of 1. Asterisks indicate a significant difference from the control (P < 0.05). (C) XBP1S enhances GEP-stimulated endochondral ossification. (a and b) Safranin O-fast green staining of metatarsals. Metatarsals were explanted from 15-day-old mouse embryos and cultured in the absence (CTR) or presence of Ad-GEP (MOI 20) with or without Ad-XBP1S. After 5 days of culture, safranin O-fast green staining was observed by using low-power (a) or high-power (b) microphotography. (c) Alizarin red S and alcian blue staining of metatarsals. The explants were fixed and processed for alizarin red S and alcian blue staining; a representative photograph is presented. (d) Per cent increase in total and mineralization length of metatarsal bones. Metatarsals were cultured as described above, the total or mineralization length was determined and the per cent increase was calculated (per cent increase = [length at day 5 − length at day 0]/length at day 0). Asterisks indicate a significant difference from the control (P < 0.05).
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fig09: XBP1S activates the chondroinductive action of granulin-epithelin precursor (GEP). (A) Schematic structures of wild-type and mutant XBP1S proteins. The N-terminal domain (N-term), one DNA binding domain (type a domain) and one transactivating domain (type b domain) are indicated. RXSXL and RXTXK are the amino acid sequences in the DNA binding domain and the transactivating domain. In the XBP1Smt-a mutant, the RXSXL in the a domain was substituted by RXGXK, in the XBP1Smt-b mutant, the RXTXK in the b domain was substituted by RXCXE, and in the XBP1Smt-a/b double mutant, the RXSXL and RXTXK motifs in both a and b domains were replaced by RXGXK and RXCXE. (B) XBP1S improves GEP-induced Col X and MMP-13 expression, while XBP1S mutants failed to enhance the GEP-induced Col X and MMP-13 expression, assayed by real-time PCR. ATDC5 cells pre-treated with BMP2 for 5 days were cultured in the absence or presence of Ad-GEP (MOI20), Ad-XBP1S (MOI20)+Ad-GEP, Ad-XBP1Smt-a (MOI20)+Ad-GEP, Ad-XBP1Smt-b (MOI 20)+Ad-GEP, Ad-XBP1Smt-a/-b (MOI 20) + Ad-GEP as indicated for an additional 3 days, and Col X and MMP-13 expression was measured by real-time PCR. The units are arbitrary, and the normalized values were calibrated against controls, here given the value of 1. Asterisks indicate a significant difference from the control (P < 0.05). (C) XBP1S enhances GEP-stimulated endochondral ossification. (a and b) Safranin O-fast green staining of metatarsals. Metatarsals were explanted from 15-day-old mouse embryos and cultured in the absence (CTR) or presence of Ad-GEP (MOI 20) with or without Ad-XBP1S. After 5 days of culture, safranin O-fast green staining was observed by using low-power (a) or high-power (b) microphotography. (c) Alizarin red S and alcian blue staining of metatarsals. The explants were fixed and processed for alizarin red S and alcian blue staining; a representative photograph is presented. (d) Per cent increase in total and mineralization length of metatarsal bones. Metatarsals were cultured as described above, the total or mineralization length was determined and the per cent increase was calculated (per cent increase = [length at day 5 − length at day 0]/length at day 0). Asterisks indicate a significant difference from the control (P < 0.05).
Mentions: Foetal mouse metatarsals were dissected from foetal GEP mice (GEP−/−, 15-day-old embryos) and cultured in DMEM (Gibco, Carlsbad, CA, USA) containing 1% heat-inactivated foetal calf serum (Invitrogen) and 100 U penicillin-streptomycin per milliliter in the absence or presence of various stimuli for 5 days, as indicated in Figures4, 8 and 9.

Bottom Line: XBP1S stimulates chondrocyte differentiation from mesenchymal stem cells in vitro and endochondral ossification ex vivo.Furthermore, XBP1S enhances GEP-stimulated chondrogenesis and endochondral bone formation.Collectively, these findings demonstrate that XBP1S, a BMP2-inducible transcription factor, positively regulates endochondral bone formation by activating GEP chondrogenic growth factor.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Genetics, Core Facility of Development Biology, Chongqing Medical University, Chongqing, China.

Show MeSH
Related in: MedlinePlus