[Show abstract][Hide abstract] ABSTRACT: Pfeiffer syndrome is a classic form of craniosynostosis that is caused by a proline-->arginine substitution at amino acid 252 (Pro252Arg) in fibroblast growth factor receptor 1 (FGFR1). Here we show that mice carrying a Pro250Arg mutation in Fgfr1, which is orthologous to the Pfeiffer syndrome mutation in humans, exhibit anterio-posteriorly shortened, laterally widened and vertically heightened neurocraniums. Analysis of the posterior and anterior frontal, sagittal and coronal sutures of early post-natal mutant mice revealed premature fusion. The sutures of mutant mice had accelerated osteoblast proliferation and increased expression of genes related to osteoblast differentiation, suggesting that bone formation at the sutures is locally increased in Pfeiffer syndrome. Of note, dramatically increased expression of core-binding transcription factor alpha subunit type 1 (Cbfa1) accompanied premature fusion, suggesting that Cbfa1 may be a downstream target of Fgf/Fgfr1 signals. This was confirmed in vitro, where we demonstrate that transfection with wild-type or mutant Fgfr1 induces Cbfa1 expression. The induced expression was also observed using Fgf ligands (Fgf2 and Fgf8). These studies provide direct genetic evidence that the Pro252Arg mutation in FGFR1 causes human Pfeiffer syndrome and uncovers a molecular mechanism in which Fgf/Fgfr1 signals regulate intramembraneous bone formation by modulating Cbfa1 expression.
Human Molecular Genetics 08/2000; 9(13):2001-8. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have generated the first mouse model of fibro-blast growth factor receptor 3 (Fgfr3) with the K644E mutation, which accurately reflects the embryonic onset of a neonatal lethal dwarfism, thanatophoric dysplasia type II (TDII). Long-bone abnormalities were identified as early as embryonic day 14, during initiation of endochondral ossification. Increased expression of PATCHED: (PTC:) was observed, independent of unaltered expression of parathyroid hormone-related peptide (PTHrP) receptor and Indian Hedgehog (IHH:), suggesting a new regulatory role for Fgfr3 in embryos. We demonstrate that the mutation enhances chondrocyte proliferation during the early embryonic skeletal development, in contrast to previous reports that showed decreased proliferation in postnatal-onset dwarf mice with activating Fgfr3 mutations. This suggests that signaling through Fgfr3 both promotes and inhibits chondrocyte proliferation, depending on the time during development. In contrast, suppressed chondrocyte differentiation was observed throughout the embryonic stages, defining decreased differentiation as the primary cause of retarded longitudinal bone growth in TDII. This model was successfully crossed with a cartilage-specific CRE: transgenic strain, excluding the lung as the primary cause of lethality.
Human Molecular Genetics 08/2000; 9(11):1603-13. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The tumor suppressor SMAD4, also known as DPC4, deleted in pancreatic cancer, is a central mediator of TGF-beta signaling. It was previously shown that mice homozygous for a null mutation of Smad4 (Smad4-/-) died prior to gastrulation displaying impaired extraembryonic membrane formation and endoderm differentiation. Here we show that Smad4+/- mice began to develop polyposis in the fundus and antrum when they were over 6 - 12 months old, and in the duodenum and cecum in older animals at a lower frequency. With increasing age, polyps in the antrum show sequential changes from hyperplasia, to dysplasia, in-situ carcinoma, and finally invasion. These alterations are initiated by a dramatic expansion of the gastric epithelium where Smad4 is expressed. However, loss of the remaining Smad4 wild-type allele was detected only in later stages of tumor progression, suggesting that haploinsufficiency of Smad4 is sufficient for tumor initiation. Our data also showed that overexpression of TGF-beta1 and Cyclin D1 was associated with increased proliferation of gastric polyps and tumors. These studies demonstrate that Smad4 functions as a tumor suppressor in the gastrointestinal tract and also provide a valuable model for screening factors that promote or prevent gastric tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: Missense mutations in fibroblast growth factor receptor 3 (FGFR3) result in several human skeletal dysplasias, including the most common form of dwarfism, achondroplasia. Here we show that a glycine-to-cysteine substitution at position 375 (Gly375Cys) in human FGFR3 causes ligand-independent dimerization and phosphorylation of FGFR3 and that the equivalent substitution at position 369 (Gly369Cys) in mouse FGFR3 causes dwarfism with features mimicking human achondroplasia. Accordingly, homozygous mice were more severely affected than heterozygotes. The resulting mutant mice exhibited macrocephaly and shortened limbs due to retarded endochondral bone growth and premature closure of cranial base synchondroses. Compared with their wild-type littermates, mutant mice growth plates shared an expanded resting zone and narrowed proliferating and hypertrophic zones, which is correlated with the activation of Stat proteins and upregulation of cell-cycle inhibitors. Reduced bone density is accompanied by increased activity of osteoclasts and upregulation of genes that are related to osteoblast differentiation, including osteopontin, osteonectin, and osteocalcin. These data reveal an essential role for FGF/FGFR3 signals in both chondrogenesis and osteogenesis during endochondral ossification.
Journal of Clinical Investigation 01/2000; 104(11):1517-25. · 12.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Missense mutations of human fibroblast growth factor receptor 3 (FGFR3) result in several skeletal dysplasias, including hypochondroplasia, achondroplasia and thanatophoric dysplasia. To study the function of FGFR3 in bone growth and to create animal models for the FGFR3-related inherited skeletal disorders, we introduced a point mutation (Lys644Glu) into the murine FGFR3 genome using a knock-in approach. We found that the Lys644Glu mutation resulted in retarded endochondral bone growth with severity directly linked to the expression level of the mutated Fgfr3. Mice heterozygous for the mutation ( Fgfr3(TD/+) ) expressed the mutant allele at approximately 20% of the wild-type level and exhibited a mild bone dysplasia. However, when the copy number of the mutant allele increased from one (Fgfr3(TD/+) to two (Fgfr3(TD/TD), the retardation of bone growth became more severe and showed phenotypes resembling those of achondroplasia patients, characterized by a dramatically reduced proliferation of growth plate cartilage, macrocephaly and shortening of the long bones, which was most pronounced in the femur. Molecular analysis revealed that expression of the mutant receptor caused the activation of Stat1, Stat5a and Stat5b, and the up-regulation of p16, p18 and p19 cell cycle inhibitors, leading to dramatic expansion of the resting zone of chondrocytes at the expense of the proliferating chondrocytes. The mutant growth plates consequently were in a less active state and generated fewer maturing and hypertrophic chondrocytes. These data provide direct genetic evidence that the point mutations in FGFR3 cause human skeletal dysplasias and uncover a mechanism through which the FGFR3 signals regulate bone growth by modulating expression of Stats and ink4 cell cycle inhibitors.
Human Molecular Genetics 02/1999; 8(1):35-44. · 7.69 Impact Factor