FGFR3 Deficiency Causes Multiple Chondroma-like Lesions by Upregulating Hedgehog Signaling

<div><p>Most cartilaginous tumors are formed during skeletal development in locations adjacent to growth plates, suggesting that they arise from disordered endochondral bone growth. Fibroblast growth factor receptor (FGFR)3 signaling plays essential roles in this process; however, the role of FGFR3 in cartilaginous tumorigenesis is not known. In this study, we found that postnatal chondrocyte-specific <i>Fgfr3</i> deletion induced multiple chondroma-like lesions, including enchondromas and osteochondromas, adjacent to disordered growth plates. The lesions showed decreased extracellular signal-regulated kinase (ERK) activity and increased Indian hedgehog (IHH) expression. The same was observed in <i>Fgfr3</i>-deficient primary chondrocytes, in which treatment with a mitogen-activated protein kinase (MEK) inhibitor increased <i>Ihh</i> expression. Importantly, treatment with an inhibitor of IHH signaling reduced the occurrence of chondroma-like lesions in <i>Fgfr3</i>-deficient mice. This is the first study reporting that the loss of <i>Fgfr3</i> function leads to the formation of chondroma-like lesions via downregulation of MEK/ERK signaling and upregulation of IHH, suggesting that FGFR3 has a tumor suppressor-like function in chondrogenesis.</p></div

Inhibition of IHH signaling suppresses growth plate expansion and the formation of chondroma-like lesions in <i>Fgfr3</i> cKO mice.

<p>(<b>A–H</b>) X-ray and micro-CT images of knee joints in Cre-negative mice treated with vehicle (n = 9) or SMOi (n = 8) and <i>Fgfr3</i> cKO mice treated with vehicle (n = 9) or SMOi (n = 8). SMOi treatment reduced knee joint deformation (white arrowheads) in <i>Fgfr3</i> cKO mice. (<b>I–P</b>) Fast Green/Safranin O staining showing a reduction in growth plate expansion and chondroma-like lesion formation (black arrowheads) in <i>Fgfr3</i> cKO mice after SMOi treatment, which resulted in premature fusion of growth plates (arrows). (<b>Q</b>) Femur and tibia length was measured from X-ray images. (<b>R</b>) Measurement of the growth plate cartilage area by histomorphometry. (<b>S</b>) Chondroma-like lesions of the femur and tibia of <i>Fgfr3</i> cKO mice treated with vehicle or SMOi, as visualized by radiographic and histological examination; lesion formation was reduced by SMOi treatment. Values represent mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. Scale bar: 200 μm.</p

FGFR3/fibroblast growth factor receptor 3 inhibits autophagy through decreasing the ATG12–ATG5 conjugate, leading to the delay of cartilage development in achondroplasia

<p>FGFR3 (fibroblast growth factor receptor 3) is a negative regulator of endochondral ossification. Gain-of-function mutations in <i>FGFR3</i> are responsible for achondroplasia, the most common genetic form of dwarfism in humans. Autophagy, an evolutionarily conserved catabolic process, maintains chondrocyte viability in the growth plate under stress conditions, such as hypoxia and nutritional deficiencies. However, the role of autophagy and its underlying molecular mechanisms in achondroplasia remain elusive. In this study, we found activated FGFR3 signaling inhibited autophagic activity in chondrocytes, both in vivo and in vitro. By employing an embryonic bone culture system, we demonstrated that treatment with autophagy inhibitor 3-MA or chloroquine led to cartilage growth retardation, which mimics the effect of activated-FGFR3 signaling on chondrogenesis. Furthermore, we found that FGFR3 interacted with ATG12–ATG5 conjugate by binding to ATG5. More intriguingly, FGFR3 signaling was found to decrease the protein level of ATG12–ATG5 conjugate. Consistently, using in vitro chondrogenic differentiation assay system, we showed that the ATG12–ATG5 conjugate was essential for the viability and differentiation of chondrocytes. Transient transfection of ATG5 partially rescued FGFR3-mediated inhibition on chondrocyte viability and differentiation. Our findings reveal that FGFR3 inhibits the autophagic activity by decreasing the ATG12–ATG5 conjugate level, which may play an essential role in the pathogenesis of achondroplasia.</p

Histological assessment of the knee and wrist in <i>Fgfr3</i> cKO mice.

<p>(<b>A</b>–<b>P</b>) Fast green/Safranin O- and H & E-stained sagittal sections of the proximal tibia of Cre-negative and <i>Fgfr3</i> cKO mice. (<b>A</b>–<b>D</b>) Images of multiple osteochondroma- and enchondroma-like lesions around disordered growth plates of the tibia in <i>Fgfr3</i> cKO but not in Cre-negative mice. Higher magnification views of areas shown in boxes in E–P. (<b>E</b>–<b>H</b>) Expansion of the region occupied by hypertrophic chondrocytes in <i>Fgfr3</i> cKO growth plates. (<b>I–L</b>) Images of enchondroma-like lesions attached to lamellar bone trabeculae in <i>Fgfr3</i> cKO mice. (<b>M–P</b>) Structure of the cartilaginous cap in <i>Fgfr3</i> cKO mice resembling growth plate cartilage. (<b>Q</b>–<b>X</b>) Fast Green/Safranin O- and H & E-stained coronal sections of the ulna and radius of Cre-negative and <i>Fgfr3</i> cKO mice. Higher magnification views of areas shown in boxes in U–X. (<b>U</b>–<b>X</b>) Images of enchondroma- (U, V) and osteochondroma-like (W, X) lesions in the ulna and radius of <i>Fgfr3</i> cKO mice. Scale bar: 1 mm (A–D, Q–T), 200 μm (E–P, U–X).</p

Impaired homeostasis of growth plate chondrocytes in <i>Fgfr3</i> cKO mice.

<p>Sections of the tibia from 8-week-old mice injected with tamoxifen for 4 weeks were analyzed by histology or immunohistochemistry. (<b>A</b>) H & E-stained sagittal sections of the proximal tibia of a Cre-negative (control) mouse showing growth plate chondrocytes organized into columns, in contrast with the expanded and disorganized growth plate of <i>Fgfr3</i> cKO mice. Dense chondrocyte clusters (upper panel, arrows) within the epiphysis region were observed in <i>Fgfr3</i> cKO mice. (<b>B</b>) PCNA- and Ki67-positive cells in the proliferative zone of Cre-negative mice; a greater number of immunoreactive cells were observed in <i>Fgfr3</i> cKO mice and were also present in the hypertrophic zone. The ratio of PCNA- to Ki67-positive cells in growth plates was calculated. Values represent mean ± SD. *p < 0.05 (n = 3). (<b>C</b>) Expansion of collagen 10 and MMP 13 expression domains in the growth plate of <i>Fgfr3</i> cKO mice. (<b>D</b>) qRT-PCR analysis of mRNA expression in primary chondrocytes from <i>Fgfr3</i> cKO and Cre-negative control mice. Data are expressed as the percent expression relative to controls. Values represent mean ± SD. *p < 0.05 vs. controls. (<b>E</b>) Apoptosis in growth plates of Cre-negative and <i>Fgfr3</i> cKO mice. Total TUNEL-positive cells (arrows) in the growth plate hypertrophic zone were counted. Values represent mean ± SD (n = 9). Scale bar: 200 μm (A, C, D, E); 100 μm (B).</p

Model of chondroma-like lesion formation in <i>Fgfr3</i> cKO mice.

<p>(<b>A</b>) Effect of <i>Fgfr3</i> deficiency on chondroma-like lesion formation. (<b>B</b>) Schematic of normal growth plate in long bone. Growth plate chondrocytes are well-organized and surrounded by the perichondrium. (<b>C</b>) Loss of <i>Fgfr3</i> in growth plate chondrocytes leads to changes in signaling and in chondrocyte polarity, proliferation, differentiation, and apoptosis, resulting in the formation of chondroma-like lesions. (<b>D</b>) Growth plate chondrocytes near the perichondrium subsequently form osteochondroma-like lesions, while those in middle region form enchondroma-like lesions. RZ, resting zone; PZ, proliferative zone; PHZ, pre-hypertrophic zone; PZ, hypertrophic zone; TB, trabecular bone; GR, groove of Ranvier.</p

Loss of <i>Fgfr3</i> suppresses ERK activation but enhances IHH expression.

<p>(<b>A</b>) IHH protein expression in growth plates was enhanced in <i>Fgfr3</i> cKO relative to Cre-negative mice, and was abundant in chondroma-like lesions of mutants, as determined by immunohistochemistry. (<b>B</b>) Phospho-ERK expression was reduced in growth plates of <i>Fgfr3</i> cKO mice. (<b>C</b>) ERK activity in response to FGF18 was impaired in <i>Fgfr3</i>-deficient chondrocytes, as determined by western blotting. (<b>D</b>) Control chondrocytes, chondrocytes treated with MEK inhibitor (250 nM U0126 or 10 nM PD98059), and <i>Fgfr3</i>-deficient chondrocytes were evaluated for <i>Ihh</i> and <i>Pthrp</i> expression by qRT-PCR. Data are expressed as the percent expression relative to controls. Values represent mean ± SD. *p < 0.05 vs. controls. Scale bars: 200 μm.</p

Gross morphology and radiographic assessment of skeletal phenotypes in <i>Fgfr3</i> cKO mice.

<p>(<b>A</b>) Gross morphology of 12-week-old <i>Fgfr3</i> cKO mice with large stature. Femur and tibia lengths were determined from X-ray images; their lengths were increased in <i>Fgfr3</i> cKO as compared to Cre-negative mice. Values represent mean ± SD. ***p < 0.001, **p < 0.01 (n = 10). (<b>B</b>) X-ray and Micro-CT images showing subluxation/dislocation of the radial head (asterisks) and arch-like deformation of the radius (arrowheads) in <i>Fgfr3</i> cKO mice. Bony lesions (arrows) in mutants were attached to the distal ulna and radius. (<b>C</b>) Gross morphology and X-ray/micro-CT images of the knee joint of 12-week-old <i>Fgfr3</i> cKO mice after skin removal showing a bony lesion (arrows) contiguous with the proximal tibia affected by knee joint deformity (arrowheads). (<b>D, E</b>) Rib bones showing aberrant radiographic density (D, arrows) and an irregular costal cartilage surface (E, arrows) were observed by X-ray and micro-CT in <i>Fgfr3</i> cKO mice.</p