Saturday, October 1, 2011


FGFR3 is like the anti-CNP.  Inducing ERK1/2-phosphorylation seems to be bad for height at least in active growth plates.  However ERK1/2 may also increase Sox9 and increase Sox9 phosphorylation.  So ERK1/2 is good for forming new growth plates.  And FGFR3 may have the ERK1/2 independent effect of increasing Stat1.

A novel FGFR3-binding peptide inhibits FGFR3 signaling and reverses the lethal phenotype of mice mimicking human thanatophoric dysplasia.

"Gain-of-function mutations in FGFR3 lead to several types of human skeletal dysplasia syndromes including Achondroplasia (ACH), Hypochondroplasia (HCH) and Thanatophoric dysplasia (TD). Currently there are no effective treatments for these skeletal dysplasia diseases. We screened, using FGFR3 as a bait, a random 12-peptide phage library and obtained 23 positive clones which share identical amino acid sequences (VSPPLTLGQLLS), named as peptide P3. This peptide had high binding specificity to the extracellular domain of FGFR3. P3 inhibited tyrosine kinase activity of FGFR3 and its typical downstream molecules, ERK/MAPK. P3 also promoted proliferation and chondrogenic differentiation of cultured ATDC5 chondrogenic cells. In addition, P3 alleviated the bone growth retardation in bone rudiments from mice mimicking human thanatophoric dysplasia type II (TDII). P3 reversed the neonatal lethality of TDII mice."

"TDI patients have curved, short femurs with or without cloverleaf skull and TDII patients have relatively longer femurs with severe cloverleaf skull"

"humans with downregulated FGFR3 activity exhibit camptodactyly, a syndrome with a tall stature, scoliosis and hearing loss"

"FGFR3 inhibits chondrocyte proliferation through Stat1 signaling by inducing the expression of cell cycle suppressor genes, such as the CDK inhibitor p21"

"FGFR3 also inhibits chondrocyte differentiation via the ERK/MAPK pathway"

"P3 inhibited the tyrosine kinase activity of FGFR3 and its downstream ERK/MAPK pathway in

"FGF2-mediated ERK1/2 phosphorylation was partially blocked by P3 at different time points after
FGF2 treatment"

"Sox9, Col2 and Col10,[all upregulated in LSJL] were all increased in cultures treated with P3 peptide when compared with non treated controls"

"Peptide P3 alleviated the bone growth retardation mainly by promoting chondrocyte differentiation in TDII mice."

"inhibition of the ERK/MAPK pathway by CNP or U0126 was also found to alleviate the skeletal phenotypes of mice mimicking human ACH or Apert syndrome resulting from gain-of-function mutation in FGFR3 or FGFR2, respectively"

"We generated transgenic mice that express a constitutively active mutant of MEK1 in chondrocytes. These mice showed a dwarf phenotype similar to achondroplasia, the most common human dwarfism, caused by activating mutations in FGFR3. These mice displayed incomplete hypertrophy of chondrocytes in the growth plates and a general delay in endochondral ossification, whereas chondrocyte proliferation was unaffected. Analysis of the cranial base in transgenic embryos showed reduced staining for collagen type X and persistent expression of Sox9 in chondrocytes. MAPK pathway inhibits hypertrophic differentiation of chondrocytes and negatively regulates bone growth without inhibiting chondrocyte proliferation. Expression of a constitutively active mutant of MEK1 in chondrocytes of Fgfr3-deficient mice inhibited skeletal overgrowth, strongly suggesting that regulation of bone growth by FGFR3 is mediated at least in part by the MAPK pathway. Although loss of Stat1 restored the reduced chondrocyte proliferation in mice expressing an achondroplasia mutant of Fgfr3, it did not rescue the reduced hypertrophic zone, the delay in formation of secondary ossification centers, and the achondroplasia-like phenotype. Fgfr3 signaling inhibits bone growth by inhibiting chondrocyte differentiation through the MAPK pathway and by inhibiting chondrocyte proliferation through Stat1."

"FGFs have been shown to activate multiple signal transduction pathways; these include Stat1, Stat3, Stat5, ERK1, ERK2, p38 mitogen-activated protein kinases (MAPKs), phospholipase C-γ, protein kinase C, Src, phosphatidylinositol 3-kinase, and Akt"

"loss of Stat1 restores the reduced chondrocyte proliferation and normal bone length in transgenic mice that overexpress FGF2 under the control of a phosphoglycerate kinase promoter"

"Up-regulation of Sox9 by FGFs is inhibited by a specific inhibitor of the MAPK pathway, strongly suggesting that Sox9 expression in chondrocytes can be regulated by the MAPK pathway. Sox9 is expressed in all chondroprogenitor cells and chondrocytes, but its expression is completely abolished in hypertrophic chondrocytes."

"MEK1 is activated by various growth factors including FGFs. MEK1 in turn phosphorylates and activates ERK1 and ERK2 MAPKs. "

"robust and prolonged phosphorylation of MEK1, ERK1, and ERK2 correlated with up-regulation of Sox9 and c-Fos"

"constitutively active MEK1 mutant in chondrocytes caused persistent expression of Sox9, which is normally down-regulated before hypertrophic differentiation. Overexpression of Sox9 inhibits hypertrophic differentiation of chondrocytes"

" up-regulation of Sox9, c-fos, and p21 was strongly inhibited by a specific inhibitor of the MAPK pathway, U0126. Together, these observations indicate that expression of Sox9, c-fos, and p21 was regulated by FGFs in chondrocytes in the absence of Stat1"<-So just inhibiting Stat1 may be best.

Note that LSJL still increased height and growth plate height despite almost assuredly increasing ERK1/2-p.

New insight on FGFR3-related chondrodysplasias molecular physiopathology revealed by human chondrocyte gene expression profiling.

"Activating mutations of the FGFR3 gene lead to craniosynostosis and multiple types of skeletal dysplasia with varying degrees of severity: thanatophoric dysplasia (TD), achondroplasia and hypochondroplasia. [We examined] whole gene expression differences occurring in primary human chondrocytes isolated from normal cartilage or pathological cartilage from TD-affected fetuses. The phenotype of the primary cells was confirmed by the high expression of chondrocytic markers. Altered expression of genes associated with many cellular processes was observed, including cell growth and proliferation, cell cycle, cell adhesion, cell motility, metabolic pathways, signal transduction, cell cycle process and cell signaling. Most of the cell cycle process genes were down-regulated and consisted of genes involved in cell cycle progression, DNA biosynthesis, spindle dynamics and cytokinesis. About eight percent of all modulated genes were found to impact extracellular matrix (ECM) structure and turnover, especially glycosaminoglycan (GAG) and proteoglycan biosynthesis and sulfation. Altogether, the gene expression analyses provide new insight into the consequences of FGFR3 mutations in cell cycle regulation, onset of pre-hypertrophic differentiation and concomitant metabolism changes. Moreover, impaired motility and ECM properties may also provide clues about growth plate disorganization."

"in vitro stimulation studies performed on rat chondrosarcoma (RCS) and primary chondrocytes using FGFs revealed decreased cell proliferation due to cell cycle arrest at G1, up-regulation of c-jun, junD, cyclin-D1, NF-KB, STAT1/3 and p21, activation of members of the pRb family, inactivation of Id1, cyclin-E-Cdk2 complex and reduced AKT phosphorylation{LSJL increases Akt phosphorylation}"

"The first "growth arrest initiation" step includes down-regulation of key cell cycle genes and some positive regulators of proliferation, and the second "growth arrest maintenance" step is characterized by Cdk inhibition, up-regulation of p21, Rb and p130 dephosphorylation and down-regulation of additional cell cycle protein genes"

"Most of the “cell cycle process” genes which were were down-regulated were also involved in cell cycle progression and checkpoints (e.g. BUB1, CDC2, CDKN2C, CCNA1, CCNB2{down in LSJL}, E2F8), DNA biosynthesis and replication (e.g. NFIA, PPP2A, RRM2), spindle and kinetochore assembly (e.g. BIRC5, CENPH{down}, KIF23, NDC80, TUBB2B) chromosome segregation and cytokinesis (e.g. CDC20, KIF2C, KIF14{down}, KIF15, KIF23, SEPT6). Conversely, genes promoting G1 progression, especially cyclin D1 and UHMK1 were up-regulated whereas p18, an inhibitor of G1 progression was down-regulated."

"p107 and p130 pocket proteins undergo dephosphorylation upon FGF stimulation"

"hyaluronan synthase 3 (HAS3) was up-regulated. Over-expression of this gene product may result in shortened high molecular weight hydrophilic hyaluronan molecules, possibly reducing ECM viscosity"

"mutations in PAPSS2, SLC26A2, gPAPP or SUMF1, all causing skeletal dysplasias"

"dwarfism, abnormal cell shape and disrupted columnar organization [occur] in mice lacking β1 integrin, ILK and Rac1"

" regulation of Rac1 and Rho/ROCK pathways may be impaired [in FGFR3 activating mutations], possibly resulting in reduced motility."

"several key components of many signaling pathways is modified in TDI primary chondrocytes, including DUSP6, MAP3K5, ZAK and MAP2K6 in ERK1/2 and p38MAPK signaling, ID2 and ID3 in BMP and TGFβ signaling, PIK3R1, PLCG2 and PLCB4 in inositol phosphate signaling, FOXM1 and MAPK8{down in LSJL} in JNK signaling, DKK1 in WNT signaling, (HIP), RAB23, IFT74 in hedgehog signaling or NKX3.2 in PTHrP and NFkB signaling."

"down-regulation of CNKSR2 in TDI chondrocytes may impair the crosstalk between MAPK and Ral pathways which is essential for proper receptor endocytosis, cytoskeleton remodeling and DNA synthesis. Likewise, down-regulation of IQGAP3 and LSP1 as well as over-expression of NME1 may modify ERK1/2 activation upon PKC or Ras activation"

"reduced JNK signaling may lead to G1 cell cycle arrest in TDI chondrocytes, as FOXM1 (−3.1 fold change) is known to promote proliferation and control the G1/S transitions by acting through MAPK8 (−2 fold change). Simultaneously, increased BMP signaling and up-regulation of FOSL2, EIF4A and RPS23 combined with ATF5 and NKX3.2 down-regulation, may induce differentiation"

To Do:

Genes upregulated in TD also up in LSJL:

Genes downregulated in TD:


"Achondroplasia is caused by a mutation in the transmembrane region of fibroblast growth factor receptor-3 (FGFR3). Wild type FGFR3 is one of many inhibitory regulators of endochondral bone growth, and following interaction with FGF ligand acts negatively on both proliferation and terminal differentiation of growth plate chondrocytes. In 98% of those with achondroplasia, the phenotype is caused by a specific point mutation in FGFR3, resulting in the substitution arginine for glycine at position 380 (Gly380Arg). This mutation is one of "gain-of-function," i.e. increased ligand-mediated signaling of FGFR3, which leads to excessive inhibition of bone growth. Similar to the treatment of rheumatoid arthritis through the systemic administration of soluble receptors for tumor necrosis factor 1, we hypothesized that systemic delivery of a soluble FGFR3 molecule would likewise titrate receptor-specific FGF ligands and thereby reduce aberrant FGFR3 signaling to rescue bone growth. Initial experiments in a murine model of achondroplasia generated by transgenic insertion of the murine orthologue of the mutant FGFR3 gene (FGFR3G374R), have shown great promise. Gene delivery of a naturally-occurring, secreted isoform of FGFR3 (FGFR3?TM) to the quadriceps of neonatal achondroplastic mice was found to provide sustained release of FGFR3?TM into the circulation. Appropriate dosing of the gene delivery vector generated levels of circulating FGFR3?TM sufficient to rescue bone growth, such that treated mice were essentially indistinguishable in size from normal littermates at four weeks. The transgenic model has been extremely useful in our proof-of-concept studies. However, certain features may unnaturally influence the amplitude of the biological response to treatment with FGFR3?TM. Therefore, to explore the treatment potential of soluble FGFR3 inhibitors in a context more relevant to the human achondroplasia mutant genotype and phenotype, we will extend these investigations to include the murine FGFR3G374R knock-in achondroplasia model. We will address issues relevant to the mechanisms supporting rescue of skeletal growth and to the potential clinical application of this treatment approach. We will address the following Specific Aims: (1) To determine the capacity of FGFR3?TM to bind FGF-ligand and thereby inhibit aberrant FGFR3G374R signaling in growth plate chondrocytes from transgenic and knock-in models of achondroplasia. (2) To determine the effects of long-term delivery of FGFR3?TM on the skeletal growth and physiology of the FGR3G374R knock-in achondroplasia model. (3) To determine the capacity of administration of a recombinant soluble FGFR3 to rescue bone growth in FGFR3G374Rneo-/+ mice. (4) To determine the relationship between age of intervention and the magnitude of the skeletal response following treatment with soluble FGFR3."

Here's the author of the grants website.  Since FGFR3 inhibits height in normal adults, this form of soluble FGFR3 may increase height in people within development.  Looking at the mentioned soluble TNF1, this will not be an easy treatment to apply.  The study scAAV-mediated gene transfer of interleukin-1-receptor antagonist to synovium and articular cartilage in large mammalian joints. mentions the involvement of gene vectors.

FGFR3 is a target of the homeobox transcription factor SHOX in limb development.

"In humans, SHOX deficiency has been associated with various short stature syndromes including Leri-Weill dyschondrosteosis (LWD), Langer mesomelic dysplasia and Turner syndrome as well as non-syndromic idiopathic short stature. A common feature of these syndromes is disproportionate short stature with a particular shortening of the forearms and lower legs. [There's] a strong positive effect of SHOX on the expression of the fibroblast growth factor receptor gene FGFR3, another well-known factor for limb development. SHOX activates the extended FGFR3 promoter, and SHOX [may directly bind] to multiple upstream sequences of FGFR3. The effect of viral overexpression of Shox in limb bud derived chicken micromass cultures was tested. Fgfr3 was negatively regulated by Shox. This repressive effect might explain the almost mutually exclusive expression patterns of Fgfr3 and Shox in embryonic chicken limbs. A negative regulation that occurs mainly in the mesomelic segments, a region where SHOX is known to be strongly expressed, offers a possible explanation for the phenotypes seen in patients with FGFR3 (e.g. achondroplasia) and SHOX defects (e.g. LWD)."

SHOX transcriptionally regulates BNP.

"The chicken Shox protein exhibits 94% homology to the human variant with 100% conservation of all known functional domains, thus suggesting functional conservation of Shox between both species."

"overexpression of Shox in chicken embryos using the replication-competent avian leukemia virus long LTR with a splice acceptor (RCAS) retroviral expression system leads to an overgrowth of the long bones in the limbs"

Genetic inhibition of fibroblast growth factor receptor 1 in knee cartilage attenuates the degeneration of articular cartilage in adult mice.

"FGFR-1 was deleted from the articular chondrocytes of adult mice in a cartilage-specific and tamoxifen-inducible manner. Two OA models (aging-associated spontaneous OA, and destabilization-induced OA), as well as an antigen-induced arthritis (AIA) model, were established and tested in Fgfr1-deficient and wild-type (WT) mice. Alterations in cartilage structure and the loss of proteoglycan were assessed in the knee joints of mice of either genotype, using these 3 arthritis models. Primary chondrocytes were isolated and the expression of key regulatory molecules was assessed quantitatively. In addition, the effect of an FGFR-1 inhibitor on human articular chondrocytes was examined.
The gross morphologic features of Fgfr1-deficient mice were comparable with those of WT mice at both the postnatal and adult stages. The articular cartilage of 12-month-old Fgfr1-deficient mice displayed greater aggrecan staining compared to 12-month-old WT mice. Fgfr1 deficiency conferred resistance to the proteoglycan loss induced by AIA and attenuated the development of cartilage destruction after surgically induced destabilization of the knee joint. The chondroprotective effect of FGFR-1 inhibition was largely associated with decreased expression of matrix metalloproteinase 13 (MMP-13) and up-regulation of FGFR-3 in mouse and human articular chondrocytes.{Both upregulation of MMP13(reduced MMP13 is associated with a form of dwarfism) and downregulation of FGFR3 have been reported to have pro-height growth effects therefore upregulation of FGFR1 may be good for height growth}"

"FGF-2 stimulates the production of matrix metalloproteinase 13 (MMP-13)"

"Fgfr3-knockout (KO) mice exhibited abnormal cartilage metabolism and early signs of OA"<-but FGFR3-KO is good for height.

"overexpression of Fgfr1 in mouse prechondrogenic ATDC5 cells led to up-regulation of Mmp13"

"FGFR-3 is down-regulated by FGF-2 via the FGFR-1/ERK/MAPK pathway in human articular chondrocytes"

A network of transcriptional and signaling events is activated by FGF to induce chondrocyte growth arrest and differentiation

" FGF inhibits chondrocyte proliferation by initiating multiple pathways that result in the induction of antiproliferative functions and the down-regulation of growth-promoting molecules. The initiation of growth arrest is characterized by the rapid dephosphorylation of the retinoblastoma protein (pRb) p107 and repression of a subset of E2F target genes by a mechanism that is independent of cyclin E–Cdk inhibition. In contrast, hypophosphorylation of pRb and p130 occur after growth arrest is first detected, and may contribute to its maintenance. Importantly, we also find a number of gene expression changes indicating that FGF promotes many aspects of hypertrophic differentiation, a notion supported by in situ analysis of developing growth plates from mice expressing an activated form of FGFR3."

"increased expression of STAT proteins, p21, and Ink family Cdk inhibitors (CDKIs) [occurs] in response to excessive FGF signaling"

"absence of both p107 and p130 impaired endochondral bone development by causing excessive proliferation and decreased differentiation of growth plate chondrocytes"

Detailed comparison of LSJL genes to genes upregulated by FGF to be done.  Spot comparison is done below.

FGF Chondrosarcoma genes upregulated at 1 hour also upregulated by LSJL:


"FGF causes changes in the expression pattern of several genes in RCS cells that are similar to those occurring during chondrocyte differentiation in vivo."

"the altered expression of these genes in the growth plates of mice harboring an activating mutation of FGFR3 mimics the changes observed in FGF-treated RCS cells, consistent with the notion that excessive FGF signaling may promote premature differentiation of chondrocytes in the mutant growth plates. "

Figure 7.
Initiation of differentiation versus maintenance of cell state.

In the supplementary data given there was a comprehensive gene list given and no alteration of Sox9 was detected.

Postnatal Soluble FGFR3 Therapy Rescues Achondroplasia Symptoms and Restores Bone Growth in Mice.

"Achondroplasia is a rare genetic disease characterized by abnormal bone development, resulting in short stature. It is caused by a single point mutation in the gene coding for fibroblast growth factor receptor 3 (FGFR3), which leads to prolonged activation upon ligand binding. To prevent excessive intracellular signaling and rescue the symptoms of achondroplasia, we have developed a recombinant protein therapeutic approach using a soluble form of human FGFR3 (sFGFR3), which acts as a decoy receptor and prevents FGF from binding to mutant FGFR3. sFGFR3 was injected subcutaneously to newborn Fgfr3(ach/+) mice-the mouse model of achondroplasia-twice per week throughout the growth period during 3 weeks. Effective maturation of growth plate chondrocytes was restored in bones of treated mice, with a dose-dependent enhancement of skeletal growth in Fgfr3(ach/+) mice. This resulted in normal stature and a significant decrease in mortality and associated complications, without any evidence of toxicity. These results describe a new approach for restoring bone growth and suggest that sFGFR3 could be a potential therapy for children with achondroplasia and related disorders. "

"sFGFR3 treatment also caused growth of the long bones in wild-type mice."

"The binding of FGF2, FGF9, or FGF18 with sFGFR3 resulted in decreased phosphorylation of FGFR and extracellular signal–regulated kinase (ERK) in chondrocytes isolated from Fgfr3ach/+ mice"

"sFGFR3 (0.25 mg/kg) was sufficient to increase body weight and length in Fgfr3ach/+ mice to match those of vehicle-treated wild-type mice, and at a dose of 2.5 mg/kg, treated dwarf mice were even heavier and had longer long bones than vehicle-treated wild-type animals."

Mice were sacrificed at day 22 of life.

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