The PI3K pathway regulates endochondral bone growth through control of hypertrophic chondrocyte differentiation
The majority of our bones develop through the process of endochondral ossification that involves chondrocyte proliferation and hypertrophic differentiation in the cartilage growth plate. A large number of growth factors and hormones have been implicated in the regulation of growth plate biology, however, less is known about the intracellular signaling pathways involved. PI3K/Akt has been identified as a major regulator of cellular proliferation, differentiation and death in multiple cell types.
Results and Discussion
Employing an organ culture system of embryonic mouse tibiae and LY294002, a pharmacological inhibitor of PI3K, we show that inhibition of the pathway results in significant growth reduction, demonstrating that PI3K is required for normal endochondral bone growth in vitro. PI3K inhibition reduces the length of the proliferating and particularly of the hypertrophic zone. Studies with organ cultures and primary chondrocytes in micromass culture show delayed hypertrophic differentiation of chondrocytes and increased apoptosis in the presence of LY294002. Surprisingly, PI3K inhibition had no strong effect on IGF1-induced bone growth, but partially blocked the anabolic effects of C-type natriuretic peptide.
Our data demonstrate an essential role of PI3K signaling in chondrocyte differentiation and as a consequence of this, in the endochondral bone growth process.
KeywordsGrowth Plate Bone Growth Alcian Blue PI3K Inhibition PI3K Pathway
Bone formation occurs through two different mechanisms: endochondral and intramembranous ossification. Longitudinal growth of the axial and appendicular skeleton is a result of endochondral ossification (EO) that is controlled by the cartilage growth plate . EO involves the aggregation of mesenchymal cells to form cartilaginous nodules . A subset of the cells in these nodules matures further into growth plate chondrocytes.
During endochondral bone development in the limb, growth plate chondrocytes undergo well-ordered and controlled phases of cell proliferation, maturation, and apoptosis . The growth plate can be divided into three main chondrocyte subpopulations: the resting, proliferating and hypertrophic chondrocytes. These populations are arranged in distinct zones that are distinguishable by morphological criteria, but are also characterized by specific molecular markers. The proliferation and/or differentiation of these subpopulations are controlled by a complex network of regulatory molecules . Proliferative chondrocytes synthesize type II collagen and form characteristic columns; they then exit the cell cycle and become post-mitotic prehypertrophic chondrocytes that differentiate further into hypertrophic cells. Hypertrophic chondrocytes express type X collagen and mineralize the surrounding matrix. This differentiation process is followed by apoptosis of hypertrophic chondrocytes, but prior to their death, they deposit vascular endothelial growth factor (VEGF) into their extracellular matrix, which promotes the invasion of blood vessels into the cartilage tissue. Blood vessel invasion enables the recruitment of osteoblasts and osteoclasts and replacement of the cartilage scaffold by a calcified bone matrix [2, 3, 4, 5]. This final step results in the formation of trabecular bone (the primary spongiosa). With continuing resorption of the primary spongiosa by osteoclasts, the primary center splits into two opposing growth plates, in each of which the maturation of cartilage and subsequent remodeling into bone continue, as long as new chondrocytes are generated in the growth plates .
Hypertrophic chondrocytes play a pivotal role in coordinating chondrogenesis and osteogenesis, as they provide a scaffold for subsequent formation of trabecular bone and secrete factors such as VEGF that control the activity of other cells involved in EO. Therefore, the proper regulation of chondrocyte differentiation and the coordination of chondrocyte progression through the cell cycle have to be tightly regulated for normal bone growth. The induction of growth arrest is a central feature of this phenotypic transition. For example, mice lacking the cyclin dependent-kinase inhibitor p57/Kip2 exhibit several developmental abnormalities including abnormal skeletogenesis . Moreover, numerous skeletal diseases are caused by deregulation of cellular proliferation and hypertrophic chondrocyte differentiation, such as a large number of skeletal dysplasias that are characterized by dwarfism, skeletal deformities, and frequently by early-onset osteoarthritis .
Both local paracrine regulators and systemic hormones control endochondral bone formation and bone remodeling throughout life. Insulin-like growth factor-I (IGF1) and C-type natriuretic peptide (CNP) are among the major stimulators of endochondral bone growth. IGF1 is the most prominent growth factor involved in linear growth regulation and it was shown to be essential for growth plate chondrocyte development. The most prominent effect of IGF1 is induction of chondrocyte hypertrophy, as shown both in IGF1 null mice and in bone cultures treated with IGF1 [9, 10, 11, 12]. In addition, studies from our lab and others identified the CNP pathway as an important anabolic regulator of endochondral bone growth [13, 14, 15]. However, the molecular and cellular mechanisms mediating the anabolic effects of both ligands are not completely understood. Substantial progress has been made in the past few years in understanding how local signaling molecules, working through key transcription factors such as Sox and Runx proteins, interact and control the growth and differentiation of bones [3, 5, 16, 17, 18]. However, the intracellular signaling pathways connecting extracellular signaling molecules to transcriptional regulators are poorly understood.
Here we focus on Phosphatidylinositol 3-kinases (PI3Ks) which represent a family of lipid kinases whose inositol lipid products are key mediators of intracellular signaling in many cell types . PI3Ks are represented by a family of eight distinct enzymes that can be divided into three classes based on their structure and function . Class I PI3Ks have been the major focus of PI3K studies because these isoforms are generally coupled to extracellular stimuli. The generation of D3-phosphorylated phosphoinositides at the membrane by PI3Ks results in the recruitment of certain signaling proteins to the plasma membrane via their pleckstrin homology (PH) domains. As such, PI3Ks are upstream regulators in a number of signaling cascades that control proliferation, growth, cell death, migration, metabolism, and a host of other biological responses . In addition, the PI3K pathway is known as the major signaling cascade downstream IGF1 in many cell types [21, 22, 23, 24].
Class I PI3Ks are reversibly inhibited by the pharmacological compound LY294002, and more specifically class I alpha isoforms by PI3-K α inhibitor IV from Calbiochem. Genetic screens in model organisms have identified Akt (protein kinase B) as the primary downstream mediator of the effects of PI3K . PtdIns (4, 5)P2 and PtdIns (3, 4, 5)P3 bind to the PH domain of Akt, recruiting the kinase to the plasma membrane where Akt is phosphorylated and activated [26, 27, 28]. Akt has been shown to be a critical mediator of cell proliferation and survival . In mice, disruption of the most ubiquitously expressed member of the Akt family of genes, Akt1, results in body size reduction compared to the wild-type littermates [30, 31].
Therefore, the PI3K/Akt pathway is involved in proliferation, differentiation, cellular survival or a combination of these processes in multiple cell types, from neurons to fibroblasts  but its role in cartilage and bone development has not been studied intensively and is the focus of our study.
LY294002 suppresses chondrocyte differentiation
PI3K inhibition results in reduced bone growth
When the three parts of the bone (gp1-proximal growth plate, gp2-distal growth plate, and min-mineralized area) were calculated as a percentage of the entire length of the bone, we noticed a relative increase in the length of the mineralized zone upon PI3K inhibition (Figure 2E).
Treatment of tibiae with LY294002 results in smaller proliferative and hypertrophic zones
Similar results were found when we calculated the length of the zones reporting the measurements as percentage of the entire growth plate, but in this case the decrease in proliferative zone length was not found to be statistically significant (Figure 3C). Since LY294002 had similar effects on the proximal or distal growth plates (Figure 2C and Figure 2D), zone measurements were performed on the proximal growth plate only.
Treatment of tibiae with LY294002 results in reduced hypertrophic cell size
To examine whether the observed effects were specific for the chosen developmental stage, we performed similar experiments with tibiae from E18.5 mice. Tibiae cultured for 6 days in the presence of LY294002 demonstrated decreased length of the growth plate and the hypertrophic zone, similar to E15.5 tibiae. We also noticed decreased size of hypertrophic cells in the presence of the PI3K inhibitor (Figure 4B), demonstrating that the anabolic role of the PI3K pathway is not specific to a certain developmental stage.
Decreased markers of chondrocyte differentiation and increased apoptosis in LY294002 treated growth plates
We next examined the effects of PI3K inhibition on chondrocyte proliferation and apoptosis. BrdU (Bromodeoxyuridine) labeling revealed no significant difference in the percentage of replicating cells within the proliferative zone of the growth plate (data not shown). Treatment of E15.5 tibiae with LY294002 or DMSO for 6 days results in increased number of cells showing positive TUNEL stain in the hypertrophic zone of LY294002 treated bones (Figure 5C).
IGF1-induced bone growth partially requires PI3K activity
C-type natriuretic peptide-induced bone growth requires PI3K activity
The PI3K pathway has been shown to affect numerous cellular processes in a tissue-specific fashion; for example, it is required for survival in different cell types such as cardiomyocytes , cellular differentiation in the case of osteoclasts and keratinocytes [35, 36], and proliferation and differentiation of osteoblasts . It also stimulates differentiation of CD4+ T-cells  and development and proliferation of B cells [38, 39]. We hypothesized that the PI3K pathway has similar effects in the growth plate, promoting endochondral bone growth by increasing proliferation and differentiation of chondrocytes and by suppressing apoptosis.
We found that inhibition of PI3K with LY294002 results in decreased differentiation, in both primary chondrocytes (micromass cultures) and organ cultures. Markers of both early chondrocyte differentiation such as collagen II and glycosaminoglycans and of late hypertrophic differentiation such as collagen X, p57, Alkaline phosphatase activity and calcium content (Alizarin red S stain) were decreased upon PI3K inhibition. These data suggest that the PI3K pathway is required for normal chondrocyte differentiation. In the organ culture system, we have shown that the PI3K pathway is required for maximal bone growth, since by inhibiting the pathway we obtained 55% reduction in bone growth, due to a proportionate shortening of both growth plates.
The major phenotype of the LY294002 treated tibiae is represented by a 45% reduction in the length of the hypertrophic zone, providing further evidence that the PI3K pathway is required for hypertrophic differentiation. The observed reduction in the area staining for collagen X and p57 in LY294002 treated tibiae is in agreement with reduced hypertrophy. In addition we have seen a 20% reduction of the length of the proliferative area of the growth plate, in LY294002 treated tibiae. In the organ culture system it seems that the onset of proliferation is delayed, since the resting zone represents a higher percentage of the growth plate in the LY294002 treated bones compared to control. The ratio of BrdU labeled cells within the proliferative zone of the growth plate does not appear to be different between LY294002 and control cultures, suggesting that PI3K inhibition results in delayed cell cycle entry, but does not affect the rate of cell cycle progression once cells have entered to proliferative zone. Our data also show increased apoptosis in organ cultures treated with LY294002. Apoptosis was only detected in the hypertrophic and mineralized zones, suggesting that the PI3K pathway is required for hypertrophic chondrocyte survival.
PI3K signaling transduces signals from many growth factors and other extracellular cues, but it is not known which of them utilizes the pathway for anabolic effects on endochondral bones. Potential candidates are IGFs, however, our data suggest, somewhat unexpectedly that IGF1 stimulates organ culture growth in the presence of LY294002 to a similar degree as in control cultures. IGF1 treatment causes an increase in the length of hypertrophic zone , and this increase is not completely blocked by the PI3K inhibitor. This suggests that the PI3K pathway is not the only and potentially not the major pathway required for IGF1-induced bone growth and -hypertrophic differentiation in our organ culture system.
One potential problem that could partially explain the lack of growth reduction in the IGF1 + LY294002 treatment is that IGF1 possibly increased Akt phosphorylation to a level that is no longer completely inhibited by 10 μM LY294002. The mechanisms for IGF1 and CNP regulation of the PI3K pathway in growth plate chondrocytes are not the focus of this manuscript, but we plan to investigate the implications of these two growth factors in more depth in future studies. It will be important to see the levels of phosphorylated Akt in all treatment combinations, by performing immunohistochemistry and Western blotting with protein isolated directly from the tibiae treated with all treatment combinations (control, IGF1, LY294002 and LY294002 + IGF1 or control, CNP, LY294002, and CNP +LY294002). In addition, future measurements of growth plate zones under all conditions might provide an explanation for the sustained anabolic effects of IGF1even in the presence of LY294002. Our results have shown that the PI3K pathway is mostly involved in hypertrophic chondrocyte differentiation, and also that the two ligands IGF1 and CNP increase the length of hypertrophic zone (Figures 6C and 7B). There were no obvious effects on the other zones, but performing growth plate zone measurements and molecular analyses (e.g. BrdU labeling) might bring additional information in the future.
It will also be of interest to determine which other pathways mediate anabolic activities of IGFs in cartilage; the other major signaling pathway implicated in IGF signaling in other cells, the MEK-ERK (mitogen-activated protein kinase kinase/extracellular regulated kinase) cascade, has been shown to suppress endochondral bone growth [33, 40, 41, 42] and is therefore an unlikely candidate for this role.
Surprisingly, C-type natriuretic peptide (CNP), which is not a known PI3K activator, was found to partially require PI3K activity to stimulate bone growth. The CNP-induced growth of cultured tibiae was blocked by the PI3K inhibitor. One interesting finding was that the effect of CNP on hypertrophy – a significant increase in the hypertrophic zone length  – was inhibited by LY294002. These data identify CNP as one signal requiring PI3K activity in cartilage, but there are other potential candidates for regulation of the PI3K pathway in endochondral bone growth, such as PTHrP (Parathyroid hormone-related protein) and integrin ligands. Studies are under way in our laboratory to identify physiological activators of PI3K signaling in cartilage.
The molecular mechanisms mediating the effects of PI3K signaling in endochondral bone growth remain to be identified. We show that Akt proteins are phosphorylated under control conditions, and that this activation is reduced under PI3K inhibition, resulting in reduced bone growth, in agreement with reduced growth in Akt1-deficient mice as well as mice deficient in multiple Akt genes[43, 44]. The PI3K/Akt pathway was shown to be involved in Runx2 (runt related transcription factor 2) -dependent osteoblast and chondrocyte differentiation in 2 cell lines, MC3T3-E1 and ATCDC5, respectively . Therefore it represents a candidate for the PI3K involvement in chondrocyte hypertrophy. Further investigations of the PI3K/Akt mechanisms of chondrocyte differentiation are necessary in order to find the direct targets of this signaling pathway.
We have shown that PI3K is required for normal growth plate chondrocyte differentiation and survival in vitro, and therefore for endochondral bone growth. Future studies are required to further analyze the mechanisms by which PI3K exerts these effects, investigating both the molecules downstream of PI3K and the upstream activators of the pathway, and the mechanisms used by these molecules in order to function within the PI3K/Akt pathway.
Timed pregnant CD1 mice were purchased from Charles River Laboratories. Cell culture and organ culture medium components and general chemicals were purchased from Sigma and Invitrogen. LY294002, PI3-K α inhibitor IV, and the TdT-FragEL™ DNA Fragmentation Detection Kit were purchased from Calbiochem, Antibodies for immunohistochemistry were purchased from Sigma (monoclonal anti-collagen type X-C7974), Cell Signaling Technology® (P-Akt (Ser 473) #4051; Akt #9272), Santa Cruz Biotechnology® (Kip2 p57 (H-91)). Hoechst 33342 nuclear acid stain was purchased from MolecularProbes; Pronase E, used for antigen retrieval in immunohistochemistry, from Sigma (#P5147) and AEC substrate-chromogen was purchased from Dako.
Mouse embryos were dissected at E11.5 and mesenchymal cells were isolated from limb buds by digestion for 90 minutes in dispase as described . Cells were resuspended in medium containing 60% F12, 10% FBS, 0.25% L-glutamine and 0.25% Penicillin-Streptomycin and plated at high density (2.5 × 107 cells/ml) in 10 μl droplets, to stimulate high density cellular contacts. The cells were cultured for up to 12 days, and the medium was supplemented with 2 μl beta-glycerophosphate (final concentration 1 mM) and 20 μl ascorbic acid (final concentration 0.25 mM) for each ml of medium. Medium was changed daily . Cells were differentiated in micromass culture for 3 days to allow chondrogenesis to occur before addition of LY294002 (10 μM) or DMSO and staining with Alcian blue or Alizarin red S and for alkaline phosphatase activity, as described [46, 47, 48]. Alcian blue-stained micromass cultures were incubated with 500 μl of 6 M Guanidine hydrochloride over night to extract the stain, as described . The absorbance of the Alcian blue solution was measured at 620 nm.
Measurement of DNA content in micromass cultures using Hoechst staining
The UV-excitable DNA stain Hoechst 33342 at final concentration of 5 μg/ml was used to quantify DNA content in the micromass cultures. Micromass cultures from the same trials used in the Alcian blue, Alizarin red and alkaline phosphatase stains, plated in parallel wells, were used for this experiment. Culture times, treatments and fixation procedures were all carried out in the same conditions as for the above mentioned stains. Cells were then incubated with Hoechst DNA dye for 15 minutes, washed with PBS and trypsinized 2 × 10 minutes at 37°C. The cells were then centrifuged at 1000 rpm for 2 minutes and re-suspended in culture medium. Re-suspended cells were used to measure the DNA content in these cultures using a fluorimeter (Model RF-M2004, Photon Technology International, London, ON) with excitation at 350 nm and emission at 450 nm. Data from 3 different trials was analyzed using Felix32 Software.
RNA isolation and real-time PCR
RNA was isolated from micromass cultures as previously described . Taqman real-time PCR was performed to quantitatively asses RNA samples [46, 48, 49] with primers and probe sets from Applied Biosystems; data were normalized to Gapdh (Glyceraldehyde 3-phosphate dehydrogenase) mRNA levels and represent averages and SD from direct comparison of LY294002 and DMSO treatments from at least 3 different trials.
Tibiae were isolated from E15.5 mice and cultured for 6 days in medium containing alpha MEM, ascorbic acid, beta-glycerophosphate, bovine serum albumine, glutamine and Penicillin-Streptomycin, as described . After dissection, the bones were incubated in this medium over night and then treated with LY294002 (10 μM), PI3-K α inhibitor IV (500 nM) or DMSO. In the case of IGF1 treatments the tibiae were cultured in the presence of control (0.1% Bovine serum albumine (BSA) in PBS with DMSO), IGF1- 50 ng/ml- (with DMSO), LY294002 (with 0.1% BSA) or IGF1+LY294002. The control for CNP was 0.1% HCl-BSA in PBS and the CNP concentration used, 10-6 M. The treatments were organized similar to IGF1. Media and supplements were changed every two days. The bone length was measured at the beginning (before any treatment) and end of the time course. After 6 days of treatment, organs were fixed and paraffin embedded. 5 μm sections were stained with Hematoxylin and Eosin (H&E), Safranin O/Fast green and Alcian blue and then analyzed using a Leica DMRA2 microscope. Whole tibiae were also stained with Alcian blue/Alizarin red S.
Histology and immunohistochemistry
Histology and immunohistochemistry procedures were performed as described  with minor modifications. Sections were incubated in 3% H2O2 for 15 min at room temperature, followed by boiling for 2 min and incubation for 30 min at 97°C in 10 mM sodium citrate (pH 6.0) or pronase E treatment (1 mg/ml of PBS) for 10 min in the case of collagen X. They were then blocked with 5% goat serum. Sections were incubated with primary antibodies over night at 4°C. The UltraVision LP Large Volume Detection System AP Polymer was then used to recognize the primary antibodies according to manufacturer's instructions. After washing, the HRP (horseradish peroxidase) conjugated polymer complex was visualized by incubation for 2–10 min with AEC (3-amino-9-ethylcarbazole) substrate-chromogen and then sections were washed and mounted. All images were taken at room temperature with a Retiga EX camera connected to a Leica DMRA2 microscope. Primary image analyses were performed using Openlab 4.0.4 software.
The bones were cultured for 6 days in the presence of LY294002 or DMSO, fixed in 4% Paraformaldehyde and paraffin embedded. 5 μm sections were used for the TUNEL assay using the Calbiochem® DNA Fragmentation Detection Kit according to manufacturer's instructions.
All data were collected from at least 3 independent trials, which were run in triplicate or quadruplicate. Data were expressed as mean ± SD, and * p values under 0.05 were considered significant (*). Statistical significance was determined by one-way ANOVA (for Figure 2B) and two-way ANOVA (for the rest of the graphs), with Bonferroni post-test using GraphPad Prism 3.00 for Windows.
V.U. and J.R.G. are recipients of graduate scholarships from the Canadian Arthritis Network; J.R.G. is also a recipient of an Ontario Graduate Scholarship (OGS) and K.H. is the recipient of summer studentships from the Natural Sciences and Engineering Research Council. F.B. is the recipient of a Canada Research Chair Award. Work in the lab of F.B. is supported by grants from the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, The Arthritis Society and the Canadian Arthritis Network.
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