|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2012) 36, 691696 (Printed in Great Britain)
Cross-talk between FGF and other cytokine signalling pathways during endochondral bone development
Min Jin, Xiaolan Du and Lin Chen1
State Key Laboratory of Trauma, Burns and Combined Injury, Trauma Center, Institute of Surgery Research, Daping Hospital, Third Military Medical University, Peoples Republic of China
FGF (fibroblast growth factor)/FGFR (FGF receptor) signalling plays an essential role in both endochondral and intramembranous bone development. FGF signalling pathways are important for the earliest stages of limb development and throughout skeletal development. The activity and the outcome of this signalling pathway during bone development are also influenced by many other intracellular and extracellular signals. In this review, we focus on the interplay between FGF signalling and other pathways, which is tightly regulated both spatially and temporally during endochondral skeletal development.
Key words: endochondral bone development, FGF, FGFR, cross-talk, signalling pathway
Abbreviations: ALP, alkaline phosphatase, BMP, bone morphogenetic protein, ERK, extracellular-signal-regulated kinase, FGF, fibroblast growth factor, FGFR, FGF receptor, hh, hedgehog, Ihh, Indian hh, MAPK, mitogen-activated protein kinase, MEK, MAPK/ERK kinase, PI3K, phosphoinositide 3-kinase, PIP3, phosphatidylinositol-3,4,5-triphosphate, PTEN, phosphatase and tensin homologue deleted on chromosome 10, PTHrP, parathyroid hormone-related peptide, Shh, sonic hh, TAK1, transforming growth factor β-activated kinase 1, TCF, T-cell transcription factor
1To whom correspondence should be addressed (email email@example.com).
The appendicular skeleton, facial bones, vertebrae and the lateral medial clavicles are formed by endochondral ossification. During endochondral ossification, mesenchymal cells first differentiate into chondrocytes to form a cartilage template, which is eventually replaced by bone (Karsenty and Wagner, 2002). Chondrogenesis is the earliest step of endochondral ossification. The process of chondrogenesis begins with mesenchymal cell recruitment, migration, proliferation and condensation (Hall and Miyake, 2000; Tuan, 2004). In the centre of the condensations, mesenchymal cells differentiate into chondrocytes, which proliferate during the elongation of the skeletal elements. Subsequently chondrocytes exit the cell cycle, differentiate into pre-hypertrophic and hypertrophic chondrocytes, produce mineralized ECM (extracellular matrix) and finally undergo apoptosis (Linsenmayer et al., 1991). The undifferentiated mesenchymal cells remaining at the periphery of the skeletal element form the perichondrium. When chondrocyte hypertrophy is initiated, perichondrial cells start to differentiate into osteoblasts, forming the periost (St-Jacques et al., 1999). The calcified matrix of hypertrophic chondrocytes favours vascular invasion from the periost, bringing in osteoclasts to degrade the calcified matrix and osteoblasts to deposit the bone matrix of the primary spongiosa (Vu et al., 1998). The ossification process proceeds and the cartilage template is converted into bone.
2. Fibroblast factor signalling
FGFs (fibroblast growth factors) comprise a family of 23 genes encoding structurally related secreted proteins (Powers et al., 2000; Ornitz and Itoh, 2001; Su et al., 2008). Six subfamilies of FGFs, grouped by gene structure and amino acid sequence similarities, tend to have similar biochemical and functional properties, and are expressed in specific spatial and developmental patterns (Chen and Deng, 2005). FGFs exert their biological activities by activating four distinct FGFRs (FGF receptors) with differential FGF-binding properties. A typical FGFR contains an extracellular ligand-binding domain, a transmembrane domain, and an intracellular divided tyrosine kinase domain. FGFs bind to the extracellular domain of FGFR and induce phosphorylation of tyrosine residues in the FGFR intracellular domain. The activated tyrosine kinase receptor recruits target proteins of the signalling cascade to its cytoplasmic tail and modifies them by phosphorylation (Powers et al., 2000). This leads to the activation of intracellular signalling pathways such as Ras to MAPK (mitogen-activated protein kinase), PI3K (phosphoinositide 3-kinase) to Akt [also known as PKB (protein kinase B); PI3K/Akt], PLC (phospholipase C) and PKC (protein kinase C) pathways (Eswarakumar et al., 2005; Su et al., 2008).
In addition to the classical FGF signalling pathways, other genes can positively or negatively regulate FGF signalling at the level of FGFRs or their downstream molecules. For example, Sef interacts with FGFRs and prevents phosphorylation of FRS2, or blocks FGF signalling downstream of MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase] (Furthauer et al., 2002; Tsang et al., 2002). Spry inhibits FGF signalling by sequestering Grb2 (growth-factor-receptor-bound protein 2), preventing its binding to FRS2 or directly interacts with Raf, playing its inhibitory role (Hanafusa et al., 2002; Christofori, 2003). XFLRT3 can positively regulate FGF signalling through interacting with FLRT3 (Bottcher et al., 2004).
3. FGF signalling in endochondral skeletal development
Activating mutations in FGFRs 1, 2 or 3 cause multiple human skeletal dysplasias, including dwarfing chondrodysplasia syndromes and craniosynostosis syndromes (Ornitz and Marie, 2002). Several mouse models mimicking the human achondroplasia phenotype have been created by expressing mutated forms of FGFR3 in the developing cartilage anlagen, which display a severe shortening of the appendicular skeletal elements due to reduced regions of proliferating and hypertrophic chondrocytes (Naski and Ornitz, 1998; Chen et al., 1999; Iwata et al., 2000, 2001). Conversely, mice carrying a targeted deletion of FGFR3 have the characteristics of increased regions of proliferating and hypertrophic chondrocytes (Colvin et al., 1996; Deng et al., 1996). A number of reports have suggested that FGF signalling inhibits proliferation and chondrocyte differentiation (Ornitz and Marie, 2002). In contrast with the generally held interpretation that FGF inhibits chondrocyte differentiation, some evidence suggests that FGF promote certain aspects of chondrocyte hypertrophic differentiation (Minina et al., 2002; Dailey et al., 2003).
FGF signalling is also an important regulator of osteogenesis. Mice lacking FGFR1, either in progenitor cells or in differentiated osteoblasts, have increased bone mass. However, inactivation of FGFR1 in osteo-chondroprogenitor cells delays osteoblast differentiation, whereas FGFR1 deficiency in differentiated osteoblasts accelerates differentiation (Jacob et al., 2006). Mice conditionally lacking FGFR2 or harbouring a mutation in the mesenchymal splice form of FGFR2 have decreased osteoblast proliferation and quiescent osteoblast morphology, but otherwise normal differentiation, leading to dwarfism and decreased bone mineral density (Eswarakumar et al., 2002; Yu et al., 2003). Thus, FGFR2 signalling positively regulates bone growth by osteoblasts. Mice lacking FGFR3 also have decreased bone mineral density and osteopenia (Xiao et al., 2004; Valverde-Franco et al., 2004).
4. Cross-talk between FGF signalling and other pathways involved in endochondral skeletal development
4.1. FGF and the BMP (bone morphogenetic protein) pathway
BMP signalling pathways are also crucial for skeletal development. There are at least 2 distinct pathways mediating BMP signalling: the canonical Smad pathway and a MAPK pathway (Derynck and Zhang, 2003). BMPs transduce signals through heteromeric complexes of type I and type II serine/threonine kinase receptors. Upon BMP binding, type II receptors phosphorylate serine/threonine residues in type I receptors. On activation, the receptor complex phosphorylates receptor-regulated Smad proteins (R-Smads), including Smad1, 5 and 8. Subsequently, activated R-Smads recruit and bind the common partner, Smad4. This Smad complex enters the nucleus, where it directly binds the defined elements on the DNA and regulates target gene expression together with numerous other factors (Derynck and Zhang, 2003; Yoon and Lyons, 2004). BMPs can also signal by activating TAK1 [TGFβ (transforming growth factor β)-activated kinase 1) or Ras/ERK1/2 or RhoA/ROCK (Rho-associated kinase) signalling. TAK1 interacts with MEKK1 (MEK kinase 1) and activates p38 and JNK cascades (Moustakas and Heldin, 2005). In the long bones, BMPs 2, 3, 4, 5 and 7 are expressed primarily in the perichondrium, and BMP7 is also expressed in proliferating chondrocytes (Minina et al., 2002). BMPR1a is highly expressed in the pre-hypertrophic and hypertrophic zones of the growth plates, whereas BMPR1b is highly expressed in the resting and pre-hypertrophic zones (Yoon et al., 2006).
In early mouse embryos, FGFs promote limb-bud outgrowth, whereas BMP-mediated inhibition of FGFs terminate this growth. Interestingly, a high-level FGF down-regulates Gremlin1, an antagonist of BMP. Thus, a negative feedback loop is set up to tune down FGF signals by indirectly activating BMP, which prevents limb-bud overgrowth (Verheyden and Sun, 2008). Throughout chondrogenesis, the rate of chondrocyte proliferation is determined by the balance of signalling by BMPs and FGFs (Minina et al., 2002). Mice lacking the receptor BMPR1a and/or BMPR1b have defective cartilage development, partly due to an elevation in FGF signalling that suppresses chondrogenesis. In the growth plate of these mutant mice, both FGFR1 protein level and Erk1/2 activity are higher than in wild-type animals, suggesting an antagonistic interaction between FGF/MAPK pathway and BMP signalling (Yoon et al., 2006). Furthermore, the functional antagonism between BMP and FGF signalling pathways has been found in limb culture studies. BMP treatment rescues the achondroplasia phenotype in a mouse model, and FGF treatment neutralizes the effects of BMPs (Minina et al., 2002).
However, it is not clear how these 2 signalling pathways regulate one another in chondrocytes. Some evidence suggests that FGFs and BMPs can mutually regulate the expression of signalling components, although the evidence is conflicting as to whether the regulation is positive or negative. Activated FGFR3 inhibits BMP4 expression in post-natal mouse growth plates (Naski and Ornitz, 1998), but induces BMP4 and BMP7 in embryonic growth plates, where BMP treatment also promotes FGF-18 expression (Minina et al., 2002). These conflicting results may be due to age differences in the mice (Minina et al., 2002). Recent studies also observe FGF signalling in chondrocytes increases BMP ligand (BMP2 and BMP7) mRNA expression and decreases BMP antagonist (Noggin) mRNA expression in a MAPK-dependent manner, suggesting a role for their interaction in increased bone formation (Matsushita et al., 2009). C-terminal Smad1/5 phosphorylation is known to be reduced by FGF stimulation and elevated by FGF inhibition in limb cultures (Retting et al., 2009). Interestingly, the interaction between BMP and FGF pathways can be both positive and negative for some target genes. FGF signalling can antagonize canonical Smad signalling in chondrocytes by activating ERK/MAPK pathways, but FGF-mediated pathways have positive effects on Msx2 promoter activity by non-ERK/MAPK pathways downstream of FGF (Retting et al., 2009). Although evidence from other systems demonstrates that the MEK1 pathway can phosphorylate the linker region of Smad1, subsequently inhibiting BMP signalling (Pera et al., 2003; Sapkota et al., 2007), levels of Smad linker phosphorylation in the growth plate may be regulated independently of FGFs, or FGF-mediated phosphorylation may require the activity of other factors (Retting et al., 2009). There is similar evidence that BMPs can inhibit the phosphorylation and activation of STAT3 (signal transducer and activator of transcription 3), but the mechanism is unknown (Kawamura et al., 2000). Whether this level of regulation accounts for the antagonism between BMP and FGF signalling in chondrocytes remains to be seen.
FGF signalling pathways interact with BMP signalling pathways synergistically during osteogenesis. The exogenous FGF application would promote BMP-induced ectopic bone formation. FGF-4/FGFR signals play an important role during rhBMP2-induced bone formation (Kubota et al., 2002). Low-dose FGF-2 facilitates BMP2-induced ectopic bone formation by altering the expression of BMPRs on the surface of bone forming progenitor cells (Nakamura et al., 2005). Low-dose administration of FGF-2 also augments rhBMP2-induced osteoinductive activity (Tanaka et al., 2006). FGF-2 treatment of developing calvaria stimulates BMP2 gene expression. Runx2 regulates the expression of BMP2 in response to FGF stimulation in cranial bone development (Choi et al., 2005). Further studies show that there is no significant increase in periosteal bone formation induced by BMP2 in FGF-2−/− mice, with no significant increase in ALP (alkaline phosphatase) positive activity in calvarial osteoblasts or ALP mineralized colonies in stromal cultures after BMP2 treatment. Moreover, BMP2-induced osteoclast formation is also impaired in marrow stromal cultures from FGF-2−/− mice. Interestingly, the nuclear accumulation of the runt-related transcription factor (Runx2) induced by BMP2 is markedly impaired in osteoblasts from FGF-2−/− mice. BMP2-induced p42/44 MAPK is also reduced in FGF-2−/− mice (Naganawa et al., 2008). The mis-expression of Noggin, an antagonist of BMPs, prevents cranial suture fusion in vitro and in vivo. FGF-2 and syndromic FGFR signalling suppress noggin expression, which suggest inappropriate down-regulation of noggin expression may result in syndromic FGFR-mediated craniosynostoses (Warren et al., 2003).
4.2. FGF and the hh (hedgehog) pathway
There are 3 hh proteins produced in mammals: Shh (sonic hh), Dhh (desert hh) and Ihh (Indian hedgehog). They bind to the membrane-bound receptor patched (Ptc) and relieve its suppression on another membrane protein called Smoothened (Smo), leading to activation of Gli transcription factors. During early stage of limb growth and patterning, Shh initiates the expression of FGF-4 in the ectoderm. The expression of Shh and FGF-4 is co-ordinately regulated by a positive feedback loop operating between the posterior mesoderm and the overlying apical ectodermal ridge (Laufer et al., 1994; Khokha et al., 2003).
Ihh is also a member of the hh family of proteins, being expressed in distal pre-hypertrophic and proximal hypertrophic chondrocytes (Bitgood and McMahon, 1995). The Ihh receptor, patched (Ptc) and associated signalling molecules (Smo, Gli1, Gli3) are located throughout the growth plate and perichondrium (Iwasaki et al., 1997). Knockout of Ihh results in reduced chondrocyte proliferation and premature differentiation (Kronenberg, 2003). PTHrP (parathyroid hormone-related peptide) is normally expressed by perichondrial cells at the end of skeletal elements, and inhibits hypertrophic differentiation by maintaining cells in the proliferative state (Lee et al., 1995). A model has been proposed in which Ihh serves as a measure for the number of chondrocytes undergoing hypertrophic differentiation, whereas the downstream factor PTHrP prevents chondrocytes from initiating the differentiation process. They interact in a negative feedback loop to control the length of the proliferative zone, and thus the extent of bone growth (Lanske et al., 1996; Vortkamp et al., 1996; St-Jacques et al., 1999). Ihh expression is reduced in dwarf mice expressing activated FGFR3 (Naski and Ornitz, 1998). Moreover, induction of RMD-1 cell differentiation by Ihh or rShh-N is synergistically enhanced by co-treatment with BMP2, but is blocked by co-treatment with FGF-2, indicating that promoted differentiation of chondrogenic precursor cells by hh proteins can be regulated by synergistic or antagonist cofactors (Enomoto-Iwamoto et al., 2000). FGF-2 inhibits bone growth in culture and induces down-regulation of Ihh and PTHrP gene expression; PTHrP partially reverses the inhibition of long bone growth caused by activation of FGFR3 (Chen et al., 2001). FGF signalling may act upstream of the Ihh/ PTHrP system in regulating the onset of hypertrophic differentiation (Minina et al., 2002). It seems that FGF signals regulate the onset of hypertrophic differentiation by directly regulating Ihh expression.
4.3. FGF and the PI3K/Akt pathway
PI3K converts PIP2 (phosphatidylinositol-4,5-bisphosphate) to PIP3 (phosphatidylinositol-3,4,5-triphosphate). PIP3 recruits and regulates a number of downstream effectors, the most important being the serine/theronine kinase Akt (PKB). The activity of PI3K is counteracted by the tumour suppressor protein, PTEN (phosphatase and tensin homologue deleted on chromosome 10), which is a lipid phosphatase that removes the phosphate group from the 3′ position of the inositol ring of PIP3, thereby blocking Akt activation (Hanahan and Weinberg, 2000; Cantley, 2002; Bader et al., 2005; Salmena et al., 2008).
PI3K/Akt signalling shows pleiotropic functions in chondrogenesis. PI3K/Akt signalling is required for the proliferation of chondrocyte cell lines and the production of sulfated GAG (glycosaminoglycan) in primary human articular chondrocytes (Oh and Chun, 2003; Starkman et al., 2005; Priore et al., 2006; Qureshi et al., 2007). Analyses of prechondrogenic ATDC5 cells have shown that a constitutively active form of Akt accelerates the insulin-dependent chondrogenic differentiation of ATDC5 cells, indicating that PI3K/Akt signalling promotes the differentiation of chondrocyte precursors into early-stage chondrocytes (Hidaka et al., 2001). In contrast, PI3K inhibitor reduces the expression of the early chondrocyte differentiation marker collagen II(a1) and the production of proteoglycan in ATDC5 cells (Fujita et al., 2004). Akt1/Akt2 double-knockout mice display a developmental delay in ossification of the bones (Peng et al., 2003). Akt1−/− Akt3+/− mice exhibit multiple defects in the thymus, heart and skin, and die within several days of birth, while Akt1+/− Akt3−/− mice survive normally. Moreover, Akt1/Akt3 double-knockout causes embryonic lethality at around embryonic days 11 and 12, with more severe developmental defects in the cardiovascular and nervous systems (Yang et al., 2005). Chondrocyte-specific PTEN knockout mice have dyschondroplasia resembling human enchondroma, with impaired chondrocyte proliferation and differentiation (Yang et al., 2008).
IGF-1 (insulin-like growth factor 1) prevents apoptosis induced by FGFR3 mutation through the PI3K pathway and MAPK pathway (Koike et al., 2003). FGFs 2 and 4 promote the proliferation of stem cell antigen-1(+) BMMSCs by activation of the ERK1/2 and PI3K/Akt signalling pathways (Choi et al., 2008). In addition, PI3K activity is increased in the immediate protective effect of FGF-2 on human osteoblastic cell apoptosis, although without induction of phosphorylation of Akt (Debiais et al., 2004). Akt phosphorylation is down-regulated after FGF treatment to chondrocytes, which only affects chondrocyte proliferation and not the ability of FGF to induce differentiation genes (Priore et al., 2006).
4.4. FGF and the Wnt pathway
Wnt signalling is mediated by the transcription co-factor, β-catenin. In the absence of Wnt, the level of cytosolic β-catenin is controlled by the so-called ‘β-catenin destruction complex’, composed of Axin, APC (adenomatous polyposis coli), GSK3 and CKIα (casein kinase 1α). β-catenin is constitutively phosphorylated by this complex and then targeted by ubiquitination, keeping the Wnt pathway in an ‘OFF’ state. When the Wnt ligand binds to its receptor, Frizzled, and co-receptor, LRP5/6, activation of the receptor inhibits the destruction complex, resulting in the accumulation of β-catenin in the cell cytoplasm through an intracellular protein, Dishevelled, followed by nuclear translocation of β-catenin. Nuclear β-catenin binds the Lef (lymphocyte enhancer factor)/TCF (T-cell transcription factor) family of transcription factors and turns the Wnt pathway on (Henderson and Fagotto, 2002; Moon et al., 2002; Malbon, 2004).
Wnt signalling is involved in the signalling loop between FGF10 signalling in mesenchyme and FGF-8 signalling in ridge. Wnt signals, including Wnt2a and Wnt2c, are among the earliest signals required to induce FGFs, such as FGF10 and FGF8 (Niswander, 2003). FGF10 induces Wnt3a expression and Wnt3a activates FGF8 expression via β-catenin, which then maintains FGF10 expression in a feedback loop (Tickle and Munsterberg, 2001). Recent studies have reported that during limb development FGF and Wnt signals act together to synergistically promote proliferation while maintaining the cells in an undifferentiated multipotent state, but act separately to determine cell lineage specification (ten Berge et al., 2008). The BMP, FGF and Wnt signalling pathways can interplay to differentially regulate programmed cell death versus chondrogenic differentiation in limb mesodermal cells (Grotewold and Ruther, 2002). However, it is still unclear how FGF and Wnt signalling pathways interact during chondrogenesis.
During osteogenesis, activating FGFR2 mutations in osteoblasts down-regulate the expression of many genes reported as targets of Wnt signalling, suggesting an antagonistic effect between Wnt signalling, which promotes osteoblast differentiation and function, and FGF signalling, which inhibits these processes (Mansukhani et al., 2005). Further studies show that FGF antagonizes Wnt signalling by inhibiting Wnt-induced transcription and suggest that multiple mechanisms, including down-regulation of TCFs and Wnt receptors, contribute to this effect (Ambrosetti et al., 2008). Genetic analyses in mice revealed that activation of β-catenin co-operated with FGFR1 to regulate mesenchymal stem cells differentiation, controlling suture closure (Maruyama et al., 2010).
The interplays between FGF signalling and each of other pathways vary depending on the specific cell type and stage of bone development, and the cross-talk between them generates cell-specific patterns of gene expression. Thus, a simple rule cannot easily be generalized to describe how FGF interacts with any other signalling cascade. A major challenge in the future will be to understand the function of FGF signalling, and how it interacts with other pathways, in different cell types and distinct aspects of skeletal development. Further studies need to be performed to address the issue of transcriptional regulation, and investigate the mechanisms by which the FGF signalling pathway interacts with other signalling pathways during skeletal development. Since FGF signalling is important for skeletogenesis and for inhibiting FGF signalling pathway that can prevent skeletal dysplasias induced by FGFR mutations (Eswarakumar et al., 2006; Perlyn et al., 2006; Shukla et al., 2007), the investigation of cross-talk between the FGF signalling pathway and other signalling pathways in regulating endochondral skeletal development may have potential therapeutic implications. The finding that cross-talk between the FGF signalling pathway and other signalling pathways in the control of skeletogenesis may offer novel therapeutic strategies for FGF signalling-related skeletal disorders.
Much of our current understanding of signalling cross-talk between FGF and other pathways during bone development is based on in vitro studies, and single-gene transgenic or knock- out animals. Defining the mechanisms of signalling cross-talk between FGF and other pathways during bone development will require double-gene, triple-gene or even multiple-gene transgenic animals for in vivo genetic studies. Using in vitro regulated gene expression systems that can induce or inhibit gene expression under control, the orchestrated interaction will be temporally and spatially identified, facilitating further investigation.
The work was supported by
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Received 24 June 2011/2 March 2012; accepted 26 April 2012
Published online 19 June 2012, doi:10.1042/CBI20110352
© The Author(s) Journal compilation © 2012 International Federation for Cell Biology