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Dominant negative Bmp5 mutation reveals key role of BMPs in skeletal response to mechanical stimulation.

Ho AM, Marker PC, Peng H, Quintero AJ, Kingsley DM, Huard J - BMC Dev. Biol. (2008)

Bottom Line: Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse.Genetic analysis shows that the mutation occurs at a site encoding the proteolytic processing sequence of the BMP5 protein and blocks proper processing of BMP5.Anatomic studies reveal that this mutation affects the formation of multiple skeletal features including several muscle-induced skeletal sites in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology and Howard Hughes Medical Institute, Beckman Center B300, Stanford University School of Medicine, Stanford, California 94305, USA. andrewho@pitt.edu

ABSTRACT

Background: Over a hundred years ago, Wolff originally observed that bone growth and remodeling are exquisitely sensitive to mechanical forces acting on the skeleton. Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse. Although the processes of mechanotransduction and functional response of bone to mechanical strain have been extensively studied, the molecular signaling mechanisms that mediate the response of bone cells to mechanical stimulation remain unclear.

Results: Here, we identify a novel germline mutation at the mouse Bone morphogenetic protein 5 (Bmp5) locus. Genetic analysis shows that the mutation occurs at a site encoding the proteolytic processing sequence of the BMP5 protein and blocks proper processing of BMP5. Anatomic studies reveal that this mutation affects the formation of multiple skeletal features including several muscle-induced skeletal sites in vivo. Biomechanical studies of osteoblasts from these anatomic sites show that the mutation inhibits the proper response of bone cells to mechanical stimulation.

Conclusion: The results from these genetic, biochemical, and biomechanical studies suggest that BMPs are required not only for skeletal patterning during embryonic development, but also for bone response and remodeling to mechanical stimulation at specific anatomic sites in the skeleton.

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Related in: MedlinePlus

Bmp5clv, a Bmp5 cleavage sequence mutation, disrupts the proteolytic processing of the BMP5 protein. (a) Schematic of the Bmp5 open reading frame showing the leader signal (LEAD), pro (PRO) and mature (MAT) domains and the putative proteolytic processing site (scissors). The G-to-A mutation (*) at nucleotide 932 of the Bmp5clv allele is predicted to destroy a Taq1 restriction site and to disrupt the first arginine residue in the putative conserved "RXXR" cleavage sequence in BMP5 (box). "1" and "2" denote positions of PCR primers used in typing the Bmp5clv allele. (b) Partial sequence traces showing the G-to-A substitution (*) in Bmp5clv mutant mice. This alteration is the only nucleotide difference in the Bmp5 coding region between wild-type (wt) and Bmp5clv mice. (c) Confirmation of mutation in genomic DNA. A 158-bp PCR product (gray arrowhead) containing the site of the Bmp5clv mutation is cleaved by Taq1 into 67- and 91-bp fragments (black arrowheads) in wild-type mice, partially cleaved in Bmp5clv/+ heterozygous mice, and not cleaved in Bmp5clv/Bmp5clv mice. (d) COS-7 cells were transfected with a mammalian expression vector (vec) or the same vector driving expression of wild-type (wt) or mutant (Bmp5clv) BMP5 protein. Secreted proteins were analyzed by Western blot with antibodies against the pro (anti-PRO) or mature (anti-MAT) domain of murine BMP5. Most of the wild-type protein expressed was in the smaller cleaved form, whereas all the detectable mutant protein was non-processed. No appreciable signal was detected by either antibody in the control cells. (e) A proposed dominant-negative mechanism for the Bmp5clv mutation. Wild-type BMP5 peptides are cleaved at the proteolytic site to form functional dimers with another wild-type copy of BMP5 (gray bar) or another related BMP (black bar). Cleavage mutants produce non-processed BMP5 peptides that bind other non-processed BMP5 peptides to form inactive homodimers or bind and sequester wild-type BMP5 or other related BMPs in defective heterodimers.
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Figure 1: Bmp5clv, a Bmp5 cleavage sequence mutation, disrupts the proteolytic processing of the BMP5 protein. (a) Schematic of the Bmp5 open reading frame showing the leader signal (LEAD), pro (PRO) and mature (MAT) domains and the putative proteolytic processing site (scissors). The G-to-A mutation (*) at nucleotide 932 of the Bmp5clv allele is predicted to destroy a Taq1 restriction site and to disrupt the first arginine residue in the putative conserved "RXXR" cleavage sequence in BMP5 (box). "1" and "2" denote positions of PCR primers used in typing the Bmp5clv allele. (b) Partial sequence traces showing the G-to-A substitution (*) in Bmp5clv mutant mice. This alteration is the only nucleotide difference in the Bmp5 coding region between wild-type (wt) and Bmp5clv mice. (c) Confirmation of mutation in genomic DNA. A 158-bp PCR product (gray arrowhead) containing the site of the Bmp5clv mutation is cleaved by Taq1 into 67- and 91-bp fragments (black arrowheads) in wild-type mice, partially cleaved in Bmp5clv/+ heterozygous mice, and not cleaved in Bmp5clv/Bmp5clv mice. (d) COS-7 cells were transfected with a mammalian expression vector (vec) or the same vector driving expression of wild-type (wt) or mutant (Bmp5clv) BMP5 protein. Secreted proteins were analyzed by Western blot with antibodies against the pro (anti-PRO) or mature (anti-MAT) domain of murine BMP5. Most of the wild-type protein expressed was in the smaller cleaved form, whereas all the detectable mutant protein was non-processed. No appreciable signal was detected by either antibody in the control cells. (e) A proposed dominant-negative mechanism for the Bmp5clv mutation. Wild-type BMP5 peptides are cleaved at the proteolytic site to form functional dimers with another wild-type copy of BMP5 (gray bar) or another related BMP (black bar). Cleavage mutants produce non-processed BMP5 peptides that bind other non-processed BMP5 peptides to form inactive homodimers or bind and sequester wild-type BMP5 or other related BMPs in defective heterodimers.

Mentions: Sequencing of this newly discovered Bmp5 mutation revealed a correctly spliced Bmp5 transcript with a G-to-A substitution at base 932 of the Bmp5 coding region (Fig. 1a, b). This change destroys a Taq1 site in the second exon (Fig. 1c), providing a simple assay for following the mutation in genetic crosses.


Dominant negative Bmp5 mutation reveals key role of BMPs in skeletal response to mechanical stimulation.

Ho AM, Marker PC, Peng H, Quintero AJ, Kingsley DM, Huard J - BMC Dev. Biol. (2008)

Bmp5clv, a Bmp5 cleavage sequence mutation, disrupts the proteolytic processing of the BMP5 protein. (a) Schematic of the Bmp5 open reading frame showing the leader signal (LEAD), pro (PRO) and mature (MAT) domains and the putative proteolytic processing site (scissors). The G-to-A mutation (*) at nucleotide 932 of the Bmp5clv allele is predicted to destroy a Taq1 restriction site and to disrupt the first arginine residue in the putative conserved "RXXR" cleavage sequence in BMP5 (box). "1" and "2" denote positions of PCR primers used in typing the Bmp5clv allele. (b) Partial sequence traces showing the G-to-A substitution (*) in Bmp5clv mutant mice. This alteration is the only nucleotide difference in the Bmp5 coding region between wild-type (wt) and Bmp5clv mice. (c) Confirmation of mutation in genomic DNA. A 158-bp PCR product (gray arrowhead) containing the site of the Bmp5clv mutation is cleaved by Taq1 into 67- and 91-bp fragments (black arrowheads) in wild-type mice, partially cleaved in Bmp5clv/+ heterozygous mice, and not cleaved in Bmp5clv/Bmp5clv mice. (d) COS-7 cells were transfected with a mammalian expression vector (vec) or the same vector driving expression of wild-type (wt) or mutant (Bmp5clv) BMP5 protein. Secreted proteins were analyzed by Western blot with antibodies against the pro (anti-PRO) or mature (anti-MAT) domain of murine BMP5. Most of the wild-type protein expressed was in the smaller cleaved form, whereas all the detectable mutant protein was non-processed. No appreciable signal was detected by either antibody in the control cells. (e) A proposed dominant-negative mechanism for the Bmp5clv mutation. Wild-type BMP5 peptides are cleaved at the proteolytic site to form functional dimers with another wild-type copy of BMP5 (gray bar) or another related BMP (black bar). Cleavage mutants produce non-processed BMP5 peptides that bind other non-processed BMP5 peptides to form inactive homodimers or bind and sequester wild-type BMP5 or other related BMPs in defective heterodimers.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335095&req=5

Figure 1: Bmp5clv, a Bmp5 cleavage sequence mutation, disrupts the proteolytic processing of the BMP5 protein. (a) Schematic of the Bmp5 open reading frame showing the leader signal (LEAD), pro (PRO) and mature (MAT) domains and the putative proteolytic processing site (scissors). The G-to-A mutation (*) at nucleotide 932 of the Bmp5clv allele is predicted to destroy a Taq1 restriction site and to disrupt the first arginine residue in the putative conserved "RXXR" cleavage sequence in BMP5 (box). "1" and "2" denote positions of PCR primers used in typing the Bmp5clv allele. (b) Partial sequence traces showing the G-to-A substitution (*) in Bmp5clv mutant mice. This alteration is the only nucleotide difference in the Bmp5 coding region between wild-type (wt) and Bmp5clv mice. (c) Confirmation of mutation in genomic DNA. A 158-bp PCR product (gray arrowhead) containing the site of the Bmp5clv mutation is cleaved by Taq1 into 67- and 91-bp fragments (black arrowheads) in wild-type mice, partially cleaved in Bmp5clv/+ heterozygous mice, and not cleaved in Bmp5clv/Bmp5clv mice. (d) COS-7 cells were transfected with a mammalian expression vector (vec) or the same vector driving expression of wild-type (wt) or mutant (Bmp5clv) BMP5 protein. Secreted proteins were analyzed by Western blot with antibodies against the pro (anti-PRO) or mature (anti-MAT) domain of murine BMP5. Most of the wild-type protein expressed was in the smaller cleaved form, whereas all the detectable mutant protein was non-processed. No appreciable signal was detected by either antibody in the control cells. (e) A proposed dominant-negative mechanism for the Bmp5clv mutation. Wild-type BMP5 peptides are cleaved at the proteolytic site to form functional dimers with another wild-type copy of BMP5 (gray bar) or another related BMP (black bar). Cleavage mutants produce non-processed BMP5 peptides that bind other non-processed BMP5 peptides to form inactive homodimers or bind and sequester wild-type BMP5 or other related BMPs in defective heterodimers.
Mentions: Sequencing of this newly discovered Bmp5 mutation revealed a correctly spliced Bmp5 transcript with a G-to-A substitution at base 932 of the Bmp5 coding region (Fig. 1a, b). This change destroys a Taq1 site in the second exon (Fig. 1c), providing a simple assay for following the mutation in genetic crosses.

Bottom Line: Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse.Genetic analysis shows that the mutation occurs at a site encoding the proteolytic processing sequence of the BMP5 protein and blocks proper processing of BMP5.Anatomic studies reveal that this mutation affects the formation of multiple skeletal features including several muscle-induced skeletal sites in vivo.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Developmental Biology and Howard Hughes Medical Institute, Beckman Center B300, Stanford University School of Medicine, Stanford, California 94305, USA. andrewho@pitt.edu

ABSTRACT

Background: Over a hundred years ago, Wolff originally observed that bone growth and remodeling are exquisitely sensitive to mechanical forces acting on the skeleton. Clinical studies have noted that the size and the strength of bone increase with weight bearing and muscular activity and decrease with bed rest and disuse. Although the processes of mechanotransduction and functional response of bone to mechanical strain have been extensively studied, the molecular signaling mechanisms that mediate the response of bone cells to mechanical stimulation remain unclear.

Results: Here, we identify a novel germline mutation at the mouse Bone morphogenetic protein 5 (Bmp5) locus. Genetic analysis shows that the mutation occurs at a site encoding the proteolytic processing sequence of the BMP5 protein and blocks proper processing of BMP5. Anatomic studies reveal that this mutation affects the formation of multiple skeletal features including several muscle-induced skeletal sites in vivo. Biomechanical studies of osteoblasts from these anatomic sites show that the mutation inhibits the proper response of bone cells to mechanical stimulation.

Conclusion: The results from these genetic, biochemical, and biomechanical studies suggest that BMPs are required not only for skeletal patterning during embryonic development, but also for bone response and remodeling to mechanical stimulation at specific anatomic sites in the skeleton.

Show MeSH
Related in: MedlinePlus