Author response: Dephosphorylation of the NPR2 guanylyl cyclase contributes to inhibition of bone growth by fibroblast growth factor
2017-11-23

Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Activating mutations in fibroblast growth factor (FGF) receptor 3 and inactivating mutations in the NPR2 guanylyl cyclase both cause severe short stature, but how these two signaling systems interact to regulate bone growth is poorly understood. Here, we show that bone elongation is increased when NPR2 cannot be dephosphorylated and thus produces more cyclic GMP. By developing an in vivo imaging system to measure cyclic GMP production in intact tibia, we show that FGF-induced dephosphorylation of NPR2 decreases its guanylyl cyclase activity in growth plate chondrocytes in living bone. The dephosphorylation requires a PPP-family phosphatase. Thus FGF signaling lowers cyclic GMP production in the growth plate, which counteracts bone elongation. These results define a new component of the signaling network by which activating mutations in the FGF receptor inhibit bone growth. https://doi.org/10.7554/eLife.31343.001 eLife digest Between birth and puberty, the bones of mammals grow drastically in length. This process is controlled by many proteins, and mutations affecting these proteins can cause bones to either be too long or too short. For example, mutations of a protein called the fibroblast growth factor receptor, or FGF for short, and a protein called NPR2, can cause similar forms of dwarfism – a condition characterized by short stature. The FGF protein controls bone growth, and people with overactive receptors for FGF suffer from a form of dwarfism known as achondroplasia, while people that lack FGF receptors have longer bones. The NPR2 protein, on the other hand, produces a molecule called cGMP, which is necessary for the bones to grow. When NPR2 is blocked, less cGMP is produced, which results in shorter limbs. Previous studies of bone cells grown in the laboratory have shown that these two proteins are linked by a chain of chemical messages. When the FGF receptor is active, phosphate molecules are removed from the NPR2 protein, which reduces the amount of GMP produced. However, until now it was not known whether this mechanism also controls growth in actual bones. Here, Shuhaibar et al. used genetically modified mice in which the phosphate group could not be removed from their NPR2 enzyme. As a result, the bones of these mice were longer than usual. Shuhaibar et al. then developed an imaging technique to examine the region in the bone were growth happens. To see whether FGF reduces the amount of cGMP produced by NPR2 in these areas, cGMP was detected with a fluorescent sensor in order to be tracked. In normal mice, the FGF receptor reduced the rate at which cGMP was produced, but in mice with mutated NPR2, this did not happen. When the cells could not remove the phosphates from NPR2, cGMP levels stayed high and the bones grew longer. These findings reveal new insights into the molecular causes of dwarfism. The next step will be to identify the enzyme responsible for removing phosphate from NPR2. Blocking its activity could help to enhance bone growth. In the future, this could lead to new drug treatments for achondroplasia. https://doi.org/10.7554/eLife.31343.002 Introduction Longitudinal growth of limbs and vertebrae depends on division and differentiation of chondrocytes within the cartilage growth plates located at each end of the growing bone (see Figure 2B), resulting in formation of a scaffold that is subsequently mineralized (Kozhemyakina et al., 2015). These processes are tightly controlled by multiple regulatory pathways. One such regulator is the natriuretic peptide-stimulated guanylyl cyclase, natriuretic peptide receptor 2 (NPR2), also known as guanylyl cyclase B, which is found in growth plate chondrocytes and promotes bone elongation (Yasoda et al., 1998; Yamashita et al., 2000; Chusho et al., 2001; Tamura et al., 2004; Bartels et al., 2004). Inactivating mutations in NPR2, which reduce cGMP, result in severe shortening of bones in mice and people, causing the condition acromesomelic dysplasia type Maroteaux (AMDM), in which height is reduced by ~30% (Maroteaux et al., 1971; Tamura et al., 2004; Bartels et al., 2004; Khan et al., 2012; Geister et al., 2013; Nakao et al., 2015). Conversely, activating mutations of NPR2 result in longer bones (Miura et al., 2012; Hannema et al., 2013; Miura et al., 2014), opposite to what is seen with the AMDM patients. Activation of the NPR2 guanylyl cyclase requires the extracellular binding of C-type natriuretic peptide (CNP) and also the phosphorylation of multiple intracellular juxtamembrane serines and threonines (Potter, 1998; Potter, 2011) (Figure 1A). Previous studies have established a close correlation between NPR2 phosphorylation and NPR2 activity (Abbey-Hosch et al., 2005; Egbert et al., 2014; Robinson et al., 2017), and if these phosphorylation sites are mutated to alanine, CNP-dependent guanylyl cyclase activity is reduced to only 6% of that of the wild type protein (Potter and Hunter, 1998). Correspondingly, if the seven serines and threonines are mutated to the phosphomimetic amino acid glutamate (NPR2-7E), the maximal velocity of the CNP-dependent guanylyl cyclase activity is the same as that of the wild type enzyme, but does not decrease in response to stimuli that dephosphorylate and inactivate the wild type protein (Yoder et al., 2012; Shuhaibar et al., 2016; Robinson et al., 2017) (Figure 1A). Our previous analysis of mice in which both copies of Npr2 were globally replaced with a sequence encoding NPR2-7E (Npr27E/7E) demonstrated that dephosphorylation of NPR2 is a physiological mediator of hormonal signaling in ovarian follicles (Shuhaibar et al., 2016). These findings led us to investigate whether the phosphorylation of NPR2 could also be a mediator of growth factor signaling in bones. Figure 1 Download asset Open asset Mutation of the seven regulatory serines and threonines of NPR2 to glutamates increases bone length. (A) Regulation of NPR2 activity by the phosphorylation state of the seven juxtamembrane serines and threonines on each NPR2 monomer. Purple dots represent phosphates on these serines and threonines. Red lines represent glutamates that are substituted for the serines and threonines. (B) Npr27E/7E mice have longer tails. Measurements were made from 17 to 19 live mice of each genotype. (C) X-rays of representative tails from mice euthanized at 18 weeks of age. (D) Increased CA5 vertebra length in Npr27E/7E mice compared with wild type, determined from x-ray measurements of tails at 18 weeks of age (10–13 mice of each genotype). (E,F) Longer femurs (E) and tibias (F) in Npr27E/7E vs wild type mice. (G) Increased body length in Npr27E/7E vs wild type mice. (H) No difference in cranial width comparing Npr27E/7E and wild type mice. For E–H, each bar indicates measurements from 10 to 31 mice that were euthanized at the indicated ages. All graphs show mean ±s.e.m. Data were analyzed by unpaired t-tests, with the Holm-Sidak correction for multiple comparisons where appropriate. T-tests rather than ANOVA were used because we were only interested in comparisons between genotypes at a given age, rather than comparisons across ages. *p≤0.05; **p≤0.01; ***p≤0.001. https://doi.org/10.7554/eLife.31343.003 Figure 1—source data 1 Numerical data for Figure 1B and D–H, listing individual measurments used to calculate the means and standard errors in the corresponding graphs. Additional statistical information is also provided. https://doi.org/10.7554/eLife.31343.004 Download elife-31343-fig1-data1-v2.xlsx Another essential regulator of bone elongation is FGF receptor 3 (FGFR3); activating mutations of FGFR3 inhibit bone growth, causing achondroplasia, in which human height is reduced by ~25% (Horton et al., 1978; Rousseau et al., 1994; Shiang et al., 1994; Naski et al., 1998; Wang et al., 1999; Lorget et al., 2012; Lee et al., 2017; Ornitz and Legeai-Mallet, 2017). Conversely, mice and people lacking functional FGFR3 have longer bones (Colvin et al., 1996; Deng et al., 1996; Makrythanasis et al., 2014). Intriguingly, the greatly reduced bone length seen with activating mutations of FGFR3, resembles that seen with inactivating mutations of NPR2, and activation of NPR2 by increasing CNP opposes the decrease in bone growth caused by activating mutations of FGFR3 (Yasoda et al., 2004; Yasoda et al., 2009; Lorget et al., 2012; Wendt et al., 2015). Likewise, the increased bone length seen with mice lacking FGFR3 resembles that seen with activating mutations of NPR2 (Miura et al., 2012; Hannema et al., 2013; Miura et al., 2014). Furthermore, studies of fibroblasts and chondrogenic cells derived from embryonic carcinomas and chondrosarcomas have indicated that FGF signaling decreases NPR2 activity (Chrisman and Garbers, 1999; Ozasa et al., 2005; Robinson et al., 2017), and that the inactivation is due to dephosphorylation of NPR2 (Robinson et al., 2017). These studies suggested to us that dephosphorylation and inactivation of NPR2 could be a mechanism by which FGF decreases elongation of bones in vivo. Here we tested this hypothesis by investigating the growth of bones in Npr27E/7E mice in which NPR2 cannot be inactivated by dephosphorylation, and found that their bones are longer than those of wild type mice. We then developed a live tissue imaging system for monitoring the guanylyl cyclase activity of NPR2 in intact growth plates from wild type and Npr27E/7E mice that express a FRET sensor for cGMP. Using this system, we found that FGF-induced dephosphorylation and inactivation of NPR2 decreases cGMP production in growth plate chondrocytes, thus contributing to FGF-dependent decreases in bone growth. Results Mice in which NPR2 cannot be dephosphorylated have longer bones Npr27E/7E mice, in which NPR2 is modified to mimic a constitutively phosphorylated protein (Shuhaibar et al., 2016) (Figure 1A), have longer bones. Measurements at 8–16 weeks of age showed that the tails, femurs, tibias, and bodies of Npr27E/7E mice were 8% to 14% longer than wild type (Figure 1B,E,F,G; Figure 1—source data 1). Differences in limb and body length were seen as early as 4 weeks (Figure 1E–G). The fifth caudal vertebra was 18% longer, as measured at 18 weeks (Figure 1C,D; Figure 1—source data 1). These results indicate that when NPR2 cannot be inactivated by dephosphorylation, endochondral bone growth is increased. The width of the cranium, which is dependent on membranous, not endochondral, bone growth was unaffected (Figure 1H; Figure 1—source data 1). The increases in body and bone length, which resulted from an inability of NPR2 to be inactivated by dephosphorylation, phenocopied those previously reported for mice in which NPR2 activity was increased by overexpression of the NPR2 agonist CNP or by an activating mutation in the NPR2 guanylyl cyclase domain (Yasoda et al., 2004; Miura et al., 2012) and in a human patient with the same activating mutation in NPR2, who was 14% taller than average at 15 years of age (Miura et al., 2012). Npr27E/7E mice have longer bones due to a direct effect of the mutations on NPR2 function in the bone itself Because NPR2 is expressed in tissues outside of the bone growth plate, including the pituitary and gastro-intestinal tract (Tamura and Garbers, 2003; Sogawa et al., 2010), we tested whether the longer bones of the Npr27E/7E mice resulted from a direct action on bone rather than an indirect action on other tissues. To do this, we cultured isolated tibia from 3 to 4 day old mice, such that we could measure growth rates in the absence of extrinsic hormonal or nutritional effects. When first dissected, Npr27E/7E and wild type tibia did not differ in length (Figure 2A–C; Figure 2—source data 1). Measurements were made by adding the lengths of the proximal and distal epiphyses and the calcified ossification center (Figure 2B). The absence of a detectable difference in length is consistent with previous studies showing no difference in body or tibia length at birth comparing mice with inactivated NPR2 and wild type mice (Tamura et al., 2004). Likewise, little or no difference in body or bone length is seen at birth of humans with inactivating NPR2 mutations (Bartels et al., 2004). These previous studies indicate that NPR2 activity is not required for prenatal bone elongation. However, when bones of newborn mice were placed in culture, the rate of bone elongation over a 6 day period was reduced if the Npr2 gene was disrupted (Tamura et al., 2004). Correspondingly, when cultured in the presence of 1 μM CNP and measured over a period of 6 days, the rate of bone elongation was greater for Npr27E/7E compared with wild type tibia (Figure 2A,D; Figure 2—source data 1). Because the growth plate is located within the epiphysis region (Figure 2B), we also measured the rate of elongation within this specific region. For the proximal epiphysis, there was a 14% greater elongation rate for Npr27E/7E compared to wild type (Figure 2E; Figure 2—source data 1). These results show that Npr27E/7E mice have longer bones due to a direct effect of the mutations on NPR2 function within the bone itself. Figure 2 Download asset Open asset Npr27E/7E mice have longer bones due to a direct effect on the bone. (A) Representative wild type and Npr27E/7E tibia from 3 to 4 day old mice, cultured in vitro for 6 days in the presence of 1 μM CNP. (B) Diagram showing the methods of measurement, and the position of the growth plates. Graphs C,D, and F depict measurements of the sum of the three indicated lengths: the proximal epiphysis (left), the calcified ossification center, and the distal epiphysis (right). Graphs E and G depict measurements of the length of the proximal epiphysis only. For graphs D-G, the elongation rate was determined by subtracting the length at day 0 from the length at day 6, and dividing by 6 days. (C) Tibia length immediately after dissection. 44 wild type and 79 Npr27E/7E bones were measured. (D,E) Rates of bone elongation in the presence of 1 μM CNP. (D) shows the elongation rate of the entire bone, and E shows the elongation rate of the proximal epiphysis. 21 wild type and 41 Npr27E/7E bones were measured. (F,G) Rates of bone elongation in the absence of added CNP. (F) shows the elongation rate of the entire bone, and G shows the elongation rate of the proximal epiphysis. 23 wild type and 38 Npr27E/7E bones were measured. Data were analyzed by unpaired t-tests. *p≤0.05; **p≤0.01. https://doi.org/10.7554/eLife.31343.005 Figure 2—source data 1 Numerical data for Figure 2C–G, listing individual measurments used to calculate the means and standard errors in the corresponding graphs. Additional statistical information is also provided. https://doi.org/10.7554/eLife.31343.006 Download elife-31343-fig2-data1-v2.xlsx Interestingly, there was no effect of the Npr27E/7E mutations on bone elongation in medium without added CNP (Figure 2F,G; Figure 2—source data 1). This result suggests CNP produced by the bones of 3–10 day old mice is not sufficient to activate NPR2, at least under our culture conditions. Measurements of cGMP production in living growth plates To establish a method to investigate the signaling mechanisms underlying the increased bone growth in Npr27E/7E mice, we developed a live imaging technique for monitoring NPR2 guanylyl cyclase activity in intact growth plates, using mice that globally express a sensor for cGMP, cGi500 (Russwurm et al., 2007; Thunemann et al., 2013; Shuhaibar et al., 2015). NPR2 activity has been previously measured using homogenates of fetal tibia (Yasoda et al., 1998) and membrane preparations from chondrocyte cell lines (Ozasa et al., 2005; Robinson et al., 2017). The availability of mice expressing a sensor for cGMP, combined with the development of a new method to visualize chondrocytes in live and intact growth plates by confocal microscopy, allowed us to measure NPR2 activity under physiological conditions and with high spatial and temporal resolution. cGi500 is comprised of cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP), linked by a cGMP-binding domain; the sensitivity of cGi500 to cGMP is based on Förster resonance energy transfer (FRET). Binding of cGMP to the linker domain causes the CFP and YFP domains to move farther apart. The proximity of CFP and YFP can be detected by imaging cells using a 440 nm laser to excite CFP; the energy emitted by CFP then excites YFP in proportion to the distance between the two fluorophores. Thus measurement of the relative intensities of the light emitted by CFP and YFP provides an indicator of the cGMP concentration. The CFP/YFP emission ratio is higher when the cGMP concentration is higher, with a half-maximal response at 500 nM cGMP, and a dynamic range of ~100 nM to ~3 μM cGMP (Russwurm et al., 2007). Tibias were removed from newborn mice expressing cGi500, and placed in culture on a Millicell membrane. After 1–2 days in culture, the growth plate was exposed by cutting away the overlying tissue (Figure 3A), thus allowing imaging and rapid exchange of solutions. A tibia was then placed in a perfusion slide (Figure 3B,C) on the stage of a confocal microscope. Excitation of CFP resulted in fluorescence in all regions of the growth plate (Figure 3D), allowing identification of chondrocytes at different stages of development, based on the shape of the cells: round (also called resting), columnar (also called proliferating), and hypertrophic (Kozhemyakina et al., 2015; Ornitz and Legeai-Mallet, 2017). Columnar cells that are beginning to increase their volume, referred to as ‘prehypertrophic’, express both NPR2 and FGFR3 (Yamashita et al., 2000; Chusho et al., 2001; de Frutos et al., 2007; Kozhemyakina et al., 2015). We used this columnar/prehypertrophic region for the FRET measurements (Figure 3E), such that FGF effects on NPR2 activity could be investigated. Figure 3 Download asset Open asset Imaging of CNP-stimulated cGMP production in chondrocytes within intact growth plates. (A) Preparation of a tibia from a newborn mouse expressing the cGi500 sensor for cGMP. (B) Tibia in a perfusion slide in which medium could be flowed across the growth plate surface while imaging. (C) Higher magnification image of the tibia in the perfusion slide. (D) Confocal image of fluorescence of the entire growth plate. (E) Higher magnification image of the columnar/prehypertrophic region that was used for measurements of cGMP (from a different tibia from that shown in D). (F,G) Time courses of CFP and YFP emission intensities and the CFP/YFP ratio, before and after perfusion of 100 nM CNP. https://doi.org/10.7554/eLife.31343.007 To measure the CNP-dependent guanylyl cyclase activity of NPR2, we first recorded baseline images of CFP and YFP emission in the absence of CNP. After establishing the baseline ratio, we perfused 100 nM CNP through the imaging chamber. 100 nM is close to the EC50 for activation of NPR2 expressed in a HEK cell line (Shuhaibar et al., 2016). In response to CNP, the CFP emission intensity increased and the YFP emission intensity decreased (Figure 3F), resulting in a higher CFP/YFP ratio (Figure 3G) and indicating an increase in cGMP due to activation of NPR2. Thus, confocal microscopy of isolated tibia expressing cGi500 allowed us to monitor NPR2 activity in the live and intact growth plate for the first time. The methods described here are useful for comparing rates of cGMP production by NPR2 under different experimental conditions as will be described below. However, they cannot be interpreted as a measure of cGMP levels in chondrocytes in an intact mouse, because CNP concentrations in the extracellular space around the cells of the growth plate in vivo are unknown. In our isolated tissue preparation, in which the tibia is sliced to expose the growth plate, CNP can diffuse between the extracellular space into the large volume of culture medium. Therefore the cGMP level in the absence of added CNP in the medium is not an indicator of the cGMP concentration in vivo. FGF reduces NPR2 guanylyl cyclase activity in the growth plate To investigate whether FGF signaling reduces NPR2 activity within growth plate chondrocytes, we incubated cGi500-expressing tibias with or without FGF18, and imaged the fluorescence of chondrocytes in the columnar/prehypertrophic region. Among the many FGF we used because it is of the two FGF that function to activate FGFR3 in the growth plate and 2015; et al., 2016). In tibias without perfusion of CNP through the imaging caused a large increase in the concentration of cGMP (Figure Figure data 1). However, if the tibias were with FGF18, the increase in cGMP was (Figure Figure data 1). These results indicate that in chondrocytes within a living growth plate, FGF signaling reduces the activity of the NPR2 guanylyl results were with or of (Figure or (Figure Figure data 1). Figure 4 with 2 see all Download asset Open asset FGF reduces NPR2 guanylyl cyclase activity in growth plate chondrocytes from wild type mice, but not in those from Npr27E/7E mice. were using tibias from newborn mice and used for imaging over the next 2 days. (A) Time of CFP/YFP intensity after 100 nM CNP was perfused across a wild type growth plate, comparing tibias that were for with a or with CNP was for the of the increase in the CFP/YFP ratio in response to CNP indicates cGMP production by NPR2. This and other graphs show mean ±s.e.m. the dephosphorylation of NPR2 in response to as demonstrated in cells (Robinson et al., 2017), and the effect on NPR2 guanylyl cyclase in wild type FGF the seven regulatory serines and threonines on each are phosphorylated by After FGF the regulatory sites are dephosphorylated by two (D) Time of CFP/YFP intensity after 100 nM CNP was perfused across an Npr27E/7E growth plate, comparing tibias that were for with a or with (E,F) NPR2 with seven glutamates on each by seven and the lack of effect of FGF on NPR2 enzyme (G) in CFP/YFP after CNP comparing data from wild type tibias with and without FGF (from and data from Npr27E/7E tibias with and without FGF (from D). were determined by subtracting the mean CFP/YFP ratio before CNP perfusion from the mean ratio after CNP Data were analyzed by unpaired t-tests, with the Holm-Sidak correction for multiple T-tests rather than ANOVA were used because we were only interested in a of comparisons that tested specific **p≤0.01. Figure data 1 Numerical data for Figures and Figure and B, listing individual measurments used to calculate the means and standard errors in the corresponding graphs. Additional statistical information is also provided. Download The baseline CFP/YFP ratio before CNP perfusion was the same with and without FGF that FGF no effect on the baseline cGMP concentration (Figure However, it was that the baseline cGMP level was than could be detected by the cGi500 we isolated regions from tibias and cultured under the same conditions that were used for cGi500 and measured cGMP using an this stage of limb development, all of the epiphysis is comprised of growth plate CNP the cGMP of the epiphysis was only of that after CNP (Figure Figure data and no effect of an with FGF was detected (Figure Figure data 1). These cGMP measurements also showed that in growth plates after CNP FGF for decreased cGMP to of the level (Figure consistent with the cGi500 Figure Download asset Open asset measurements of cGMP of epiphyses of tibias from wild type newborn mice. Tibias were and cultured as described for the cGi500 The proximal epiphyses were then to expose the growth plate (see Figure and the tibias were in a of medium 1 with or without for After the the epiphysis regions were and for cGMP measurement as described in the Materials and the was by a 15 in the same but also including 100 nM CNP, the conditions used for the cGi500 (A) cGMP of the epiphyses to protein The shows the mean ±s.e.m. of measurements from three (B) The from A for the no added CNP condition are with an Data were analyzed by unpaired t-tests, with the Holm-Sidak correction for multiple **p≤0.01. Figure data 1 Numerical data for Figure listing individual measurments used to calculate the means and standard errors in the corresponding graphs. Additional statistical information is also provided. Download The FGF-induced decrease in guanylyl cyclase activity is caused by NPR2 dephosphorylation of the chondrocyte cell line have shown that FGF signaling decreases NPR2 phosphorylation and and that the decrease in activity depends on the decrease in phosphorylation (Robinson et al., 2017). we have not in establishing methods to measure the phosphorylation state of NPR2 from growth plate and thus have not been to whether the dephosphorylation seen in the chondrocyte cell line also in vivo. However, we were to the Npr27E/7E mice to whether FGF reduces the guanylyl cyclase activity of NPR2 in growth plate chondrocytes by NPR2, as in Figure As with wild type, growth plates from Npr27E/7E mice showed an increase in cGMP when exposed to CNP (Figure Figure data 1). In the CNP-stimulated cGMP increase was greater for Npr27E/7E vs wild type (Figure Figure data as be if NPR2 in the wild type growth plate was dephosphorylated as a result of signaling by or growth in Figure and The higher NPR2 activity measured in the Npr27E/7E vs wild type growth plate was not by a difference in the amount of NPR2 protein, which was similar for the growth plate region of the two genotypes (Figure Another of the higher NPR2 activity for the Npr27E/7E compared to wild type is that the enzyme has a for than the enzyme, at the concentrations found in intact tissues the difference in enzyme activity be The mutations also have no effect on the CNP concentration required to activate NPR2 to its (Shuhaibar et al., 2016). in growth plates from Npr27E/7E mice, FGF did not cause a decrease in CNP-stimulated cGMP production (Figure This lack of response to FGF with the FGF-induced decrease in NPR2 activity that is seen with wild type (Figure and indicates that because the NPR2-7E protein cannot be FGF signaling does not reduce its enzyme activity (Figure These results the that FGF signaling reduces cGMP production by NPR2 in the growth plate. A PPP-family the FGF-induced decrease in NPR2 activity Our previous studies of NPR2 activity in ovarian follicles indicated that a PPP-family the dephosphorylation of NPR2 in response to et al., 2014). To if a PPP-family also the FGF-induced dephosphorylation of NPR2 in the growth plate, we tibias from newborn mice with the