1 Department of Microbiology, NYU School of Medicine, New York, NY 10016, USA
2 Department of Pathology, NYU School of Medicine, New York, NY 10016, USA
3 Department of Pharmaceutical Sciences, University of Montana, Missoula, MT 59812, USA
*Author for correspondence (e-mail: basilc01{at}med.nyu.edu)
Accepted March 13, 2001
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SUMMARY |
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Key words: FGF, Bone development, STAT1, Apoptosis, Mouse, Chondrodysplasia
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INTRODUCTION |
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In line with the genetic evidence indicating that excessive FGF signaling retards long bone growth, we previously showed (Sahni et al., 1999) that FGF signaling in chondrocytes resulted in inhibition of proliferation. Furthermore, we have also shown that treatment of chondrocytes with FGF induces phosphorylation of STAT1, a phenomenon that appears to be chondrocyte and perhaps FGFR3 specific. We have also shown, through the use of chondrocytes from Stat1-null mice, that STAT1 function is required to mediate the inhibitory effect of FGF on chondrocyte proliferation (Sahni et al., 1999). STAT1, originally identified as a signal transducing molecule in the interferon (IFN) pathway, is activated by tyrosine phosphorylation to dimerize and translocate into the nucleus where it then functions as a transcriptional regulator. STAT1 activity has previously been shown to be associated with antiviral and antiproliferative effects, most of which could be ascribed to its role as an essential mediator of IFN responses (Darnell, 1997; Levy, 1999). Thus, our findings were the first example of a physiologically relevant link between STAT1 function and a different signaling system.
As our studies indicated that STAT1 is a negative downstream regulator of chondrocyte proliferation (Sahni et al., 1999), it was expected that Stat1-/- mice would display unregulated bone growth. However, previous studies on Stat1-null mice did not reveal any gross skeletal phenotype (Durbin et al., 1996; Meraz et al., 1996). Therefore, to address the question of whether the role of STAT1 in mediating the FGF response that we observed in culture was also manifested in vivo, we crossed Stat1-/- mice with a mouse transgenic line overexpressing FGF2 (TgFGF). Adult TgFGF mice show major defects in the long bones and in the cranial bones (Coffin et al., 1995). The long bone phenotype is very similar to the chondrodysplasia described in humans with activating mutations in FGFR3. We, thus, studied the phenotype of these crosses to determine the relevance of STAT1 to FGF signaling in vivo.
We now show that the dwarfism phenotype of TgFGF mice is accompanied by impaired proliferation and increased cell death of chondrocytes in the growth plate. Both of these FGF-induced phenotypes are substantially corrected in a Stat1-/- background. Analysis of the long bone developmental program, in wild-type and Stat1-/- mice, revealed that the growth plate of Stat1-/- mice is characterized by an expansion of the proliferation zone and reduced apoptosis. These phenotypes are confined to the early postnatal development, and attenuate at adult stages. Our data demonstrate that under both physiological and unregulated FGF signaling, STAT1 function plays a crucial role in regulating the proliferation and apoptosis of the chondrocytes of the growth plate during early bone development.
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MATERIALS AND METHODS |
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Genotyping
Stat1 genotyping was performed by allele-specific PCR assay on tail DNA, using the primers: 5'-TAA TGT TTC ATA GTT GGA TAT CAT-3' (neo-resistance gene specific), 5'-CTG ATC CAG GCA GGC GTT G-3', 5'-GAG ATA ATT CAC AAAATC AGA GAG-3'. Reaction conditions were 30 cycles of 1 minute of denaturation at 94°C, 1 minute of annealing at 52°C and 2 minutes of elongation at 72°C. The PCR products were 150 bp and 320 bp for wild-type and mutant Stat1 alleles, respectively. Presence of the FGF2 transgene was confirmed by genomic PCR as described previously (Coffin et al., 1995).
Organ and cell culture
Embryonic metatarsal rudiments from E15.5 wild type, Stat1-/- , TgFGF and TgFGF/Stat1-/- were isolated and cultured as previously described (Sahni et al., 1999). Metatarsals were maintained in organ-culture for 48 hours and labeled with 50 µg/ml of BrdU solution for the last 6 hours of the culture. Bone rudiments were fixed in 4% paraformaldehyde, paraffin embedded, and cut in 5 µm sections. Bone sections were stained with anti-BrdU monoclonal antibody (Boehringer Mannheim) and a Vectastain Elite ABC Kit was used to stain cells according the manufacturers manual. Primary chondrocytes were isolated from 10-day-old growth plates and cultured, at equal density, for 24 hours, then labeled with BrdU for the last 6 hours of the experiment. The cells were fixed and stained with Hoechst and with TUNEL using in situ cell death detection kit according the manufacturers methods (Boehringer Mannheim). Cells were examined under a fluorescence microscope.
Histology and immunohistochemistry
Mice from the four genotypes described above were injected with BrdU 100 µg/g bodyweight at various ages then sacrificed after 2 hours. Femurs, tibia and calvarial bones were dissected and fixed in 4% paraformaldehyde after decalcification. The bones were embedded in paraffin and 5 µm sections were cut. Bone sections were either stained with anti-BrdU antibody, anti-Collagen X antibody, with Toluidine Blue, or with Hematoxylin and Eosin. The staining with TUNEL was performed using a POD converter followed by DAB staining as described above. The sections were counterstained with Alcian Blue.
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RESULTS |
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Radiographic analysis of the long bones of 3-month-old mice (Fig. 1A) showed that the length of both femurs and tibias were significantly reduced in TgFGF mice, consistent with previously published data (Coffin et al., 1995). Interestingly, although at 3 months of age no obvious differences were observed between wild-type and Stat1-/- bones, the crosses between TgFGF and Stat1-/- (TgFGF/Stat1-/-) showed a significant correction of the dwarfism phenotype observed in TgFGF hemizygotes (Fig. 1A,B). These results suggest that the absence of STAT1 can attenuate the inhibitory effect on bone growth produced by the unregulated expression of the FGF2 transgene. At 3 months the epiphyseal growth plate of murine bones shows significantly decreased chondrocytic turnover, and therefore the reduction of the growth plate could mask any effect that STAT1 might have on chondrocyte proliferation and/or differentiation at earlier ages. Thus, to determine the role of STAT1 in regulating FGF signaling in developing long bone, we studied the effect of Stat1 deletion on the progression of chondrocyte proliferation and differentiation during early stages of development ranging from embryonic day 17 (E17) to postnatal day 20 (P20).
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To address the role of Stat1 in mediating FGF-induced apoptosis in chondrocytes further, we performed studies in culture. Primary chondrocytes were isolated from femurs and tibias growth plates and, after 24 hours in culture, were labeled with BrdU for 6 hours, fixed and stained either with anti-BrdU antibody or with TUNEL and Hoechst to visualize the nuclei. Cells that were maintained in culture for 24 hours showed a decreased rate of DNA synthesis in TgFGF, and higher levels of proliferation in Stat1-/- compared with wild type (data not shown). The expression of the FGF2 transgene induced a significant amount of apoptosis in proliferating chondrocytes compared with the wild-type and Stat1-/- mice in which apoptosis was almost undetectable. The absence of STAT1 repressed the apoptotic effect of TgFGF (Fig. 4A,B). Accordingly, treatment of wild-type cells with exogenous FGF1 (2 ng/ml) for 24 hours led to a considerable increase in the number of apoptotic cells, but this effect was much less pronounced in Stat1-/- chondrocytes (not shown).
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Apoptosis in the calvarial bones of TgFGF mice is not corrected with the lack of STAT1
To address the specificity of the FGF/STAT1 pathway in regulating cell death, we studied the effect of excessive FGF signaling on cell death in osteoblasts during intramembranous ossification of calvarial bones. We have recently demonstrated that FGF signaling enhances apoptosis in the osteoblasts of the calvarial post-frontal suture (PF sutures) of TgFGF mice (Mansukani et al., 2000), but we could not detect STAT1 activation in this system. To address the role of STAT1 in FGF-induced apoptosis in the calvarial osteoblasts, calvaria were isolated from P1 and P10 mice that represented the four genotypes described above. Serial sections performed along the PF sutures were analyzed for the presence of apoptotic cells. At early stages (P1) no significant difference in the rate of cell death was detected between wild-type, Stat1-/-, TgFGF and TgFGF/Stat1-/- mice (not shown). However, P10 TgFGF mice exhibited high levels of apoptosis in the PF sutures compared with both wild-type and Stat1-/- mice, where the levels of cell death were low (Fig. 5A). Interestingly, when TgFGF were crossed with Stat1-/- (TgFGF/Stat1-/-) the rate of cell death remains similar to that observed in TgFGF mice. This suggests that, in contrast to growth plate chondrocytes, FGF-mediated apoptosis in osteoblasts is independent of the STAT1 pathway. Histological sections of calvarial bone (Fig. 5B) showed that in contrast to wild-type and Stat1-/- calvaria, where the PF sutures are near closure, the PF suture in TgFGF and TgFGF Stat1-/- remained wide open. Thus, in addition to STAT1-independent apoptosis, the Stat1 deletion also failed to correct the phenotype observed in calvarial sutures of TgFGF mice.
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To determine the role of STAT1 in regulating bone development in the presence and absence of unregulated FGF signaling, we performed a detailed histological analysis of the development of the growth plate. This demonstrated a general expansion of the chondrocyte population in the reserve zone, as well as in the proliferating and hypertrophic zones in the developing bones of Stat1-null mice. At P5, the reserve zone of Stat1-/- femurs was larger than that of the wild type and of the transgenics, a phenotype that was also observed in the TgFGF/Stat1-/- femurs (Fig. 6A). The reserve zones of both Stat1-/- and TgFGF/Stat1-/- mice also showed some evidence of premature chondrocyte hypertrophy, a prelude to the formation of the secondary center of ossification (Fig. 6A). The progression of chondrocytes from the reserve zone towards the proliferating zone appeared to be accelerated in Stat1-/- mice, which showed an increase in the length of the proliferating zone compared with the other three genotypes. In TgFGF mice, the reduced proliferating zone was restored to normal by the loss of STAT1. The differentiation of proliferating chondrocytes to hypertrophy leads to accumulation of hypertrophic chondrocytes, that is also enhanced in the Stat1-/- growth plates. In contrast, in TgFGF mice the hypertrophic zone is dramatically reduced and this phenotype is not corrected by the absence of STAT1 (Fig. 6A).
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To quantitate the histological observations of the developing growth plates shown in Fig. 6, we measured the height of the proliferating zone and the hypertrophic and prehypertrophic zone containing collagen type X-positive cells. As shown in Fig. 7A, between E17 and P5 there is a significant increase in the height of Stat1-/- proliferating zone compared with the wild type. These differences attenuate, as the Stat1 mutant mice become older (P10-P20). However, in TgFGF mice, the height of the proliferating zone is significantly reduced at all ages analyzed. Absence of STAT1 in TgFGF mice (TgFGF/Stat1-/-) leads to a significant increase of the proliferating zone.
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Thus, the results presented in this section show that Stat1-/- mice exhibit an early bone phenotype, characterized by an expansion of the growth plate proliferative zone and a temporary acceleration of bone growth, in line with the observation that Stat1-null growth plate chondrocytes exhibited slightly higher DNA synthesis and reduced apoptosis compared with the wild type. These alterations in bone development, however, disappeared in older animals. Conversely, TgFGF mice showed a reduction in the height of the proliferative zone, with no indication of a further block in differentiation, and shorter bones. TgFGF/Stat1-/- mice showed a partial restoration of the height of the proliferative zone and complete restoration of bone length.
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DISCUSSION |
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The chondrodysplasia observed in transgenic mice overexpressing FGF2 results from reduced proliferation and increased apoptosis of growth plate chondrocytes
For our studies we used a mouse model of achondroplasia that consists of mice overexpressing a human FGF2 transgene under the control of the PGK promoter. As previously described (Coffin et al., 1995), these mice overexpress FGF2 ubiquitously but the major phenotypic alterations consist of dwarfism, owing to chondrodysplasia and shortening of long bones and vertebrae, and also of cranial malformations that consist of delayed closing of the PF sutures and macrocephaly. Comparison of the long bones and chondrocytes of TgFGF mice with the wild type showed that chondrocyte proliferation was decreased in the reserve as well as in the proliferative zone of the growth plates. This is in line with what has been previously observed in other mouse models of FGF-related chondrodysplasias (Segev et al., 2000; Naski et al., 1998; Li et al., 1999). Unexpectedly, we also found that the chondrocytes of the growth plate of these mice exhibited a very high level of apoptosis that was detectable in vivo as well as in primary culture of TgFGF chondrocytes. Apoptosis was drastically increased in the reserve and proliferative zones of the growth plates, but was also somewhat elevated in the hypertrophic zone, where apoptosis is a normal prelude to mineralization and bone deposition. Thus, TgFGF chondrocytes show two distinct abnormalities: decreased proliferation capacity and increased apoptosis.
We had previously reported that we could not detect increased apoptosis in rat chondrosarcoma (RCS) cells after FGF treatment (Sahni et al., 1999). It is likely that the failure to observe apoptosis in these cell lines resulted from the fact that RCS derive from a chondrosarcoma, and thus, like many other tumor cells, may have developed an anti-apoptotic phenotype. Clearly it does not derive from a different effect of exogenous versus endogenous FGF, as we could easily detect increased apoptosis in FGF-treated wild-type primary chondrocytes. However, this finding implies that inhibition of proliferation is a distinct phenomenon from apoptosis, even if both the processes appear to require STAT1 (see below).
Whether the induction of apoptosis caused by unregulated FGF signaling is direct or indirect is not clear at the moment. Increased apoptosis has been reported to occur in human chondrocytes isolated from individuals with TDI in culture (Legeai-Mallet et al., 1998), which suggests that apoptosis results from the direct action of FGF on chondrocytes, rather than being dependent on the induction of expression of pro-apoptotic molecules by FGF in other cell types. However, it is possible that FGF treatment induces the production of pro-apoptotic molecules (e.g. FAS ligand) in chondrocytes, which would, in turn, be the real effector of apoptosis. It has been shown that excessive FGF signaling decreases the expression of Indian hedgehog (IHH) and parathyroid hormone-related peptide (PTHrP) receptor in the growth plate (Naski et al., 1998), and this downregulation of PTHrP signaling could also play a role in promoting apoptosis, by decreasing Bcl2 expression (Amling et al., 1997).
In conclusion, examination of various aspects of long bone development in the TgFGF mice shows that they have reduced chondrocyte proliferation and increased apoptosis, phenomena that could both contribute to defective bone growth. Apoptosis is likely to be induced directly by FGF in chondrocytes, but the major mediators of FGF-induced apoptosis have not been identified. We examined the levels of expression of various molecules that are known to regulate apoptosis in transgenic as well as in FGF-treated wild-type primary chondrocytes, but we could not detect any significant difference in the expression of Bcl2, Bax, Casp3 and Casp8. Expression of Bcl-XL, an anti-apoptotic molecule, was somewhat decreased in TgFGF chondrocytes, but the significance of this observation is not clear at this time.
The TgFGF chondrodysplasia phenotype is corrected in a Stat1-null background
By crossing the TgFGF mice with Stat1-/- mice, we obtained TgFGF/Stat1-/- mice and compared their bone phenotype with that of transgenic, Stat1+/+ or Stat1-/- littermates. As shown in the Results, the chondrodysplasia observed in TgFGF mice is substantially corrected when the FGF2 transgene is expressed in a Stat1-null background. The femurs and tibia of adult TgFGF/Stat1-/- mice are longer and less thick than those of their TgFGF littermates. Furthermore, the rate of chondrocyte proliferation in the growth plate is restored to normal or near normal levels in TgFGF/Stat1-/-. The high levels of chondrocyte apoptosis observed in growth plate chondrocytes are also considerably reduced in the crosses at all ages examined. In the hypertrophic zone, where apoptosis is a normal event and the increase in TgFGF bones is much less dramatic than in the reserve and proliferative zone, the effect of STAT1 deficiency was not very pronounced, suggesting that STAT1 does not play an important role in physiological chondrocyte apoptosis.
Stat1 was originally characterized as an essential mediator of responses to IFN, including inhibition of cell proliferation (Schindler and Darnell, 1995; Darnell, 1997). In addition, it has been implicated in the regulation of apoptosis, both in the IFN system and independently (Levy, 1999). Loss of STAT1 function in mice results in increased proliferation and decreased apoptosis of lymphocytes and correlates with decreased expression of Casp1 and Casp11, suggesting that STAT1 is required to regulate these functions (Lee et al., 2000). Stat1-mutant human fibroblasts display reduced susceptibility to tumor necrosis factor (TNF)-induced apoptosis and express reduced levels of pro-apoptotic proteins, and STAT1 has been suggested to play an adaptor function that links the TNF receptor to induction of apoptosis (Kumar et al., 1997; Wang et al., 2000). All of these mechanisms could play a role in determining the apoptotic response of chondrocytes to FGF signaling, and its modulation by STAT1. Although the precise mechanism by which STAT1 mediates FGF-induced apoptosis in chondrocytes remains to be determined, our results show that this is not a generalized effect of STAT1 deficiency on apoptosis. When we examined the calvarial bones of TgFGF and TgFGF/Stat1-/- littermates, we found that, as we previously described, osteoblasts in and around the PF suture exhibited a higher degree of apoptosis in the TgFGF skulls (Mansukhani et al., 2000). This high level of apoptosis is substantially unchanged in a Stat1-/- background. It is also noteworthy that the cranial deformities observed in the TgFGF mice are also not corrected. Thus STAT1 does not seem to mediate the FGF effects on intramembranous ossification, and indeed we could not detect STAT1 activation after FGF treatment of osteoblasts.
In conclusion, these results indicate that STAT1 is an essential downstream modulator of FGF signaling in chondrocytes in vivo and that it mediates the two major FGF effects observed in the transgenic FGF2 mice: inhibition of chondrocyte proliferation and induction of premature apoptosis. These results prompted us to study, in detail, the bone phenotype of Stat1-/- mice.
Stat1-/- mice show accelerated bone development in embryonic and early postnatal life
When we compared E17 metatarsal bone rudiments of Stat1-/- embryos with those of wild-type embryos, we noticed that Stat1-/- metatarsals were invariably slightly longer than those of their wild-type littermates. The number of DNA synthesizing cells in the growth plate was also higher, and a consistent reduction in the number of apoptotic cells was also detected. A similar trend was also apparent in 1-day-old to 10-day-old mice. The slightly increased proliferation and decreased apoptosis of Stat1-null chondrocytes produced bones in which the proliferative zone was expanded compared with that of wild-type mice, and the distance between the two opposing growth plates was also higher, thus resulting in longer bones. However, by P10-P15, those differences had essentially disappeared. This phenotype is reminiscent of that observed in Mmp9-null mice, which show a delayed apoptosis and ossification of the growth plate that is abnormally lengthened up to 3 weeks after birth. After this period, these abnormalities disappear and ultimately result in a normal axial skeleton (Vu et al., 1998).
The observation that Stat1-/- bones grow faster than the wild type is in agreement with our findings showing a profound effect of STAT1 on FGF-induced inhibition of proliferation and apoptosis. Chondrocytes produce small but detectable levels of FGFs, notably FGF2 and FGF9 (R. Kamijo, M. S. and C. B., unpublished), and the absence of STAT1 could relieve the homeostatic response to these growth regulators. The disappearance of this phenotype at later ages is more difficult to explain. As Fgfr3-null mice show longer bones than the wild type, it is unlikely that cessation of FGF production after day 10-15 renders STAT1 irrelevant to the cellular response to FGF. Rather it appears that the potential for extended growth of Stat1-null chondrocytes is limited in time by other factors or by a built-in clock that regulates the extent of chondrocyte proliferation. Alternatively, the window during which FGFR3 signaling acts through STAT1 may be limited in time, and other signal transduction pathways may produce, at later stages, a similar growth inhibition in chondrocytes. It is also possible that the effect of FGFR3 deletion results from the lack of FGF growth inhibitory signals, as well as from concomitant modulation of expression of other proteins, such as PTHrP or IHH which play a crucial role in the homeostasis of bone growth (Karp et al., 2000). Indeed PTHrP is known to stimulate the proliferation of chondrocytes and delay their differentiation, since ablation of the gene for PTHrP (Pthlh Mouse Genome Informatics) causes accelerated endochondral ossification (Karaplis et al., 1994; Chung et al., 1998; Lanske et al., 1999), while PTHrP overexpression lead to the opposite phenotype (Weir et al., 1996). PTHrP upregulation has been observed in Fgfr3-/- mice (Deng et al., 1996), and could produce an additive effect on chondrocyte proliferation. FGF induced downregulation of PTHrP expression may not require STAT1 function and thus the additive effect of PTHrP upregulation on chondrocyte proliferation would be absent in Stat1-/- mice. Experiments are in progress to determine whether this hypothesis is correct.
In conclusion, the results presented in this report show that STAT1 functions as a downstream mediator of FGF signaling during endochondral ossification in vivo, modulating the increased apoptosis and reduced chondrocyte proliferation induced by unregulated FGF signaling. The role that STAT1 plays in bone development under physiological conditions is more subtle and further experiments will be necessary to understand why it appears to be limited to early stages of bone growth.
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ACKNOWLEDGMENTS |
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REFERENCES |
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Amling, M., Neff, L., Tanaka, S., Inoue, D., Kuida, K., Weir, E., Philbrick, W. M., Broadus, A. E. and Baron, R. (1997). Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development. J. Cell Biol. 136, 205-213.
Chen, L., Adar, R., Yang, X., Monsonego, E. O., Li, C., Hauschka, P. V., Yayon, A. and Deng, C.-X. (1999). Gly369Cys mutation in mouse FGFR3 causes achondroplasia by affecting both chondrogenesis and osteogenesis. J. Clin. Invest. 104,1517-1525.
Chung, U-I., Lanske, B., Lee, K., Li, E. and Kronenberg, H. (1998). The parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation. Proc. Natl. Acad. Sci. USA 95, 13030-13035.
Coffin, J. D., Florkiewicz, R. Z., Neumann, J., Mort-Hopkins, T., Dorn II, G. W., Lightfoot, P., German, R., Howles, P. N., Kier, A., OToole, B. A. et al. (1995). Abnormal bone growth and selective translational regulation in basic fibroblast growth factor (FGF-2) transgenic mice. Mol. Biol. Cell 6, 1861-1873.[Abstract]
Colvin, J.S., Bohne, B. A., Harding, G. W., McEwen, D. G. and Ornitz, D. M. (1996). Skeletal overgrowth and deafness in mice lacking fibroblast growth factor receptor 3. Nat. Genet. 12, 390-397.[Medline]
Darnell, J.E., Jr (1997). STATs and gene regulation. Science 277, 1630-16335.
Deng, C., Wynshaw-Boris, A., Zhou, F., Kuo, A. and Leder, P. (1996). Fibroblast growth factor receptor 3 is a negative regulator of bone growth. Cell 84, 911-921.[Medline]
Durbin, J.E., Hackenmiller, R., Simon, M. C. and Levy, D. E. (1996). Targeted disruption of the mouse Stat1 gene results in compromised innate immunity to viral disease. Cell 84, 443-450.[Medline]
Gibson, G. (1998). Active role of chondrocyte apoptosis in endochondral ossification. Microsc. Res. Tech. 43, 191-204.[Medline]
Gilbert, S. F. (1994). Osteogenesis, the development of bone. In Developmental Biology. 4th edn. Chapter 9, pp. 333-338. Sunderland, MA: Sinauer Associates.
Goldfarb, M. (1996). Functions of fibroblast growth factors in vertebrate development. Cytokines Growth Factor Rev. 7, 311-325.[Medline]
Gorlin, R. J. (1997). Fibroblast growth factors, their receptors and receptor disorders. J. Cranio-Maxiollofacial Surg. 25, 69-78.
Karaplis, A. C., Luz, A., Glowacki, J., Bronson, R. T., Tybulewicz, V. L., Kronenberg, H. M. and Mulligan, R. C. (1994). Lethal skeletal dysplasia from targeted disruption of the parathyroid hormone-related peptide gene. Genes Dev. 8, 277-289.[Abstract]
Karp, S. J., Schipiani, E., St-Jacques, B., Hunzelman, J., Kronenberg, H. and McMahon, A. P. (2000). Indian Hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein-dependent and -independent pathways. Development 127, 543-548.
Kumar, A., Commane, M., Flicklinger, T. W., Horvath, C. M. and Stark, G. R. (1997). defective TNF--induced apoptosis in STAT1-null cells due to low constitutive levels of caspases. Science 278, 1630-1632.
Lanske, B., Amling, M., Neff, L., Guiducci, J., Baron, R. and Kronenberg, H. M. (1999). ablation of the PTHrP gene or the PTH/PTHrP receptor gene leads to distinct abnormalities in bone development. J. Clin. Invest. 104, 399-407.
Lee, C. K., Smith, E., Gimeno, R., Gertner, R. and Levy, D. E. (2000). STAT1 affects lymphocyte survival and proliferation partially independent of its role down-stream of IFN-gamma. J. Immunol. 164, 1286-1292.
Legeai-Mallet, L., Benoist-Lasselin, C., Delezoide, A.-L., Munnich, A. and Bonaventure. J. (1998). Fibroblast growth factor receptor 3 mutations promote apoptosis but do not alter chondrocyte proliferation in thanatophoric dysplasia. J. Biol. Chem. 273, 13007-13014.
Levy, D. E. (1999). Physiological significance of STAT proteins, investigations through gene disruption in vivo. Cell. Mol. Life Sci. 55, 1559-1567.[Medline]
Li, C., Chen, L., Iwata, T., Kitagawa, M., Fu, X.-Y. and Deng, C.-X. (1999). A Lys644Glu substitution in fibroblast growth factor receptor 3 (FGFR3) causes dwarfism in mice by activation of STATs and ink4 cell cycle inhibitors. Hum. Mol. Genet. 8, 35-44.
Mansukhani, A., Bellosta, P., Sahni, M. and Basilico, C. (2000). Signaling by fibroblastgrowth factors (FGF) and fibroblast growth factor receptor 2 (FGF2)-activating mutations blocks mineralization and induces apoptosis in osteoblasts. J. Cell Biol. 149, 1297-1308.
Meraz, M. A., White, J. M., Sheehan, K. C., Bach, E. A., Rodig, S. J., Dighe, A. S., Kaplan, D. H., Riley, J. K., Greenlund, A. C., Campbell, D. et al. (1996). Targeted disruption of the Stat1 gene in mice reveals unexpected physiologic specificity in the JAK-STAT signaling pathway. Cell 84, 431-432.[Medline]
Naski, M. C. and Ornitz, D. M. (1998). FGF Signaling in skeletal development. Front. Biosci. 3, 781-794.
Naski, M. C., Colvin, J. S., Coffin, J. D. and Ornitz, D. M. (1998). Repression of hedgehogsignaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development 125, 4977-4988.
Roach, H. I. and Clarke, N. M. (1999). Cell paralysis as an intermediate stage in the programmed cell death of epiphyseal chondrocytes during development. J. Bone Miner. Res. 14, 1367-1378.[Medline]
Rousseau, F., Bonaventure, J., Legeal-Mallet, L., Pelet, A., Rozet, J.-M., Maroteaux, P., Le Merrer, M. and Munnich, A. (1994). Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia. Nature 371, 252-254.[Medline]
Sahni, M., Ambrosetti, D.-C., Manuskhani, A., Gertner, R., Levy, D. and Basilico, C. (1999). FGF signaling inhibits chondrocyte proliferation and regulates bone development through the STAT-1 pathway. Genes Dev. 13, 1361-1366.
Segev, O., Chumakov, I., Nevo, Z., Givol, D., Madar-Shapiro, L., Sheinin, Y., Weinreb, M. and Yayon, A. (2000). Restrained chondrocyte proliferation and maturation with abnormal growth plate vascularization and ossification in human FGFR-3(G380R) transgenic mice. Hum. Mol. Genet. 9, 249-258.
Schindler, C. and Darnell, J. E. (1995). Transcriptional responses to peptide ligands, The Jak-STAT pathway. Annu. Rev. Biochem. 64, 621-651.[Medline]
Shiang, R., Thompson, L. M., Zhu, Y.-Z., Church, D. M., Fielder, T. J., Bocian, M., Winkour, S. T. and Wasmuth, J. J. (1994). Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 78, 335-342.[Medline]
Tavormina, P. L., Shiang, R., Thompson, L. M., Zhu, Y.-Z., Wilkin, D. J., Lachman, R. S., Wilcox, W. R. Rimoin, D. L., Cohn, D. H. and Wasmuth, J. (1995). Thanatophoric dysplasia (types I and II). caused by distinct mutations in fibroblast growth factor receptor 3. Nat. Genet. 9, 321-328.[Medline]
Vu, T. H., Shipley, M., Bergers, G., Berger, J. E., Helms, J. A., Hanahan, D., Shapiro, S. D., Senior, R. M. and Werb, Z. (1998). MMP-9/Gelatinase B is a key regulator of growth plate angiogenesis and apoptosis of hypertrophic chondrocytes. Cell 93, 411-422.[Medline]
Wang, Y., Wu, T. R., Cai, S., Welte, T. and Chin, Y. E. (2000). Stat1 as a component of tumor necrosis factor alpha receptor 1-TRADD signaling complex to inhibit NF-kappaB activation. Mol. Cell. Biol. 20, 4505-4512.
Webster, M. K. and Donoghue, D. J. (1997). FGFR activation in skeletal disorders, too much of a good thing. Trends Genet. 13, 178-182.[Medline]
Weir, E. C., Philbrick, W. M., Amling, M, Neff, L. A., Baron, R. and Broadus, A. E. (1996). Targeted overexpression of parathyroid hormone-related peptide in chondrocytes causes chondrodysplasia and delayed endochondral bone formation. Proc. Natl. Acad. Sci. USA 93, 10240-10245.