Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF-induced calcium signals in human diploid fibroblasts
Mutations causing achondroplasia and thanatophoric dysplasia alter bFGF-induced calcium signals in human diploid fibroblastsH. Bryant Nguyen, Mark Estacion and J. Jay Gargus*
Department of Physiology and Biophysics and Section of Human Genetics, Department of Pediatrics, University of California, Irvine, Irvine, CA 92697-4560, USA
Received November 21, 1996;Revised and Accepted February 21, 1997
Mutations in the fibroblast growth factor receptor (FGFR) gene family recently have been shown to underlie several hereditary disorders of bone development, with specific FGFR3 mutations causing achondroplasia (Ach) and thanatophoric dysplasia (TD). However, for none of these mutations has the defect in receptor function been demonstrated directly and, therefore, for none has the pathophysiological mechanism of the disease been defined. Using our established techniques for single-cell ratiometric real-time calcium image analysis, we defined the nature of the basic fibroblast growth factor (bFGF)-induced calcium signal in human diploid fibroblasts, and, in blinded studies, have analyzed the bFGF-induced signals from 18 independent fibroblast cell lines, including multiple lines from patients with known mutant alleles of FGFR3 and syndromes of Ach or TD. Control cells responded with transient increases in intracellular calcium, with many cells showing oscillatory calcium waves. Homozygous Ach cell lines failed to signal, whereas heterozygous Ach lines responded nearly normally. We observed heterogeneous signals in TD heterozygotes: the unresponsive lines all turned out to carry TD1alleles, whereas all responsive lines had TD2 alleles. Since FGFR1, 2 and 3 receptors are known to be expressed in fibroblasts, our results suggest that specific mutant FGFR3 alleles can function in a dosage-dependent dominant-negative fashion to inactivate FGFR signaling.
Mutations in the fibroblast growth factor receptor (FGFR) gene family recently have been shown to underlie several dominantly inherited disorders of bone development. FGFR1mutations have been shown to produce Pfeiffer syndrome, FGFR2 mutations have been shown to produce Crouzon, Jackson-Weiss, Pfeiffer and Apert syndrome, andFGFR3 mutations have been shown to cause achondroplasia (Ach), thanatophoric dysplasia (TD) types 1 and 2, hypochondroplasia, Crouzon syndrome with acanthosis nigricans and FGFR3-associated coronal synostosis syndrome (1 ,2 ; reviewed in 3 ,4 ). All of the mutant disease-causing alleles found in the three receptor types are dominant, mutations at different FGFR loci can give the same disease phenotype, a given allele can produce different disease phenotypes and the majority of the different alleles even appear to alter a common structure, the receptor dimer. This suggests that mutations in all three types of FGFR share a common pathophysiological mechanism. However, for none of these mutations has the defect in receptor function been demonstrated directly.
A number of groups have taken simplifying indirect approaches to approximate the pathophysiology of the FGFR mutations. These studies have entailed either the use of conventional knockout mice (5 ,6 ) or in vitro studies of transfected chimeric receptors (7 -10 ). Such studies allow one to sample different aspects of physiological FGFR signaling, but neither fully recapitulates the native or disease state. It has long been recognized that most tissues express multiple FGFR types, including splice variants of each, that most FGF ligands discriminate poorly between the receptor types and that both hetero- and homoreceptor dimers contribute to signaling (11 ). Engineered dominant-negative alleles have provided the best demonstration of this interaction, and have proven useful tools in the analysis of FGFR function (12 -14 ). Model dominant-negative mutations in one receptor class can create a dosage-dependent inhibition of all FGF signaling, even though many wild-type FGF receptors of the same and different type are expressed. This kind of interaction between the FGFR types has been defined in transfected cultured cells (12 ), injected Xenopus oocytes (12 ) and embryos (13 ), and the epidermis of transgenic mice (14 ). Since, to date, attempts to define the pathogenesis of the FGFR diseases have been limited to an analysis of either null alleles in vivo or expression of only the targeted mutant receptor itself in isolation in vitro, there has not yet been the potential to observe physiological signaling interactions between the host of receptors, expressed by most tissues, that are capable of responding in concert to the presence of FGF ligand. Since neither the knockout mice nor the transfectant systems have the potential to observe this type of physiological interaction, neither has had the potential to examine this well understood dominant-negative pathophysiological mechanism (15 ).
FGFR signaling has been studied most extensively in the model systems of the Xenopus oocyte and several cultured cell lines (12 ,16 and reviewed in 11 ). In both systems, FGF-induced intracellular calcium waves have been a reproducible part of the physiological signal, and such calcium signals increasingly have been recognized to be central and integrating components of the signals produced by the two major classes of transmembrane receptors that couple through the RAF and MAP kinases, the growth factor receptor tyrosine kinases and the G protein-coupled serpentine receptors (17 ,18 ). While the molecular nature of the membrane proteins involved in calcium signaling, and the exact functional consequences of these signals, remain open active areas of research, the fundamental membrane events underlying the generation of calcium waves themselves are already well defined (19 ).
For all of the above reasons, we felt that it would be valuable to assess directly FGF-induced calcium signals in the physiological preparation of cultured diploid fibroblasts natively expressing defined mutant FGFR3 alleles. We carried out an examination of FGF2/basic fibroblast growth factor (bFGF)-induced intracellular calcium signals in 18 independent human fibroblast cell lines, blinded to their genotype. These cells, originally obtained from affected patients, were known to be expressing specific mutant alleles, since RT-PCR of mRNA from these very cells led to the original definition of the disease gene in the laboratory of Dr J. J. Wasmuth (20 ,21 ). In addition, they should present a better model of the disease process than those models used to date since they not only physiologically express FGFR3 from genomic DNA (20 ,21 ), meaning all splice variants can be naturally produced, but they, like most tissues (11 ), also express FGFR1 and FGFR2, and the spectrum of downstream signaling proteins with which these receptors physiologically interact.
The new generation of calcium-indicator fluorescent dyes have revolutionized the measurement of intracellular calcium signaling in living cells (22 ). Uncharged esterified precursor dye passively equilibrates across the cell membrane, becoming loaded into intact cells because of intracellular esterase activity that releases charged unesterified active dye, trapping it in the cytoplasmic compartment. All cells of every shape and size are loaded simultaneously without needing microinjection or membrane damage. Furthermore the dyes' spectral properties allow for the calibrated conversion of dye signal into an intracellular calcium concentration while compensating for differences in cell thickness or dye photobleaching. This is accomplished by ratioing the dye signal recorded at two different wavelengths. Today's high sensitivity video cameras and rapid image processors allow one to perform this ratiometric operation for each pixel element of a video image of a field of cells. Thus, multiple single cells or even subcellular regions of cells in the field of view can be visualized, analyzed and measured simultaneously. A rapid repetitive resampling of a given field over time reconstructs the intracellular calcium signal from each element in the field as it responds to extracellular stimuli (22 ). The ability to examine single cells in this detail has uncovered a richness of signaling phenotypes which are invisible in an integrated population average. This is because some stimuli, such as growth factors, do not trigger responses in every cell, and because those cells that do respond show a variable latency, signal pattern and phase. Empirically, cells have a robust response to growth factors when serum deprived (23 ), therefore we chose as our standard conditions the response of serum-deprived human diploid fibroblasts to the addition of 50 ng/ml bFGF in Ringer solution, a supramaximal dose for triggering intracellular calcium waves (data not shown).
As illustrated in Figure 1 , during the first minute of baseline, agonist-free measurement, cells have a stable resting cytosolic ionized free calcium concentration of between 100 and 200 nM. Upon the addition of bFGF (indicated by the first arrow in the figure), but after a variable time lag ranging from 15 s (the time required for a 5-fold volume exchange of the bath perfusion system) to 180 s, individual cells respond asynchronously with calcium wave signals. This asynchronous response, with each cell's calcium oscillating out-of-phase with that of its neighbors, makes these signals very difficult to resolve without sophisticated single-cell analysis. In addition, the individual signals varied in the nature of the calcium wave which developed. We broadly categorized these as single transients, sustained plateaux or oscillations in the intracellular calcium concentration. The transients (Fig. 1 D) typically have a duration of 1-2 min. They are indistinguishable from the first calcium wave seen in cells that go on to oscillate (Fig. 1 A, B and E), and may, therefore, reflect a special case of the same response. Should oscillations ensue, the calcium waves occur at regular intervals, 2-3 min between peaks, and the amplitude of the waves diminishes with time. The sustained plateau response (Fig. 1 C) appears distinctly different, and may, in fact, be superimposed upon the oscillations (Fig. 1 F). It may be slow to rise, but has as its distinctive feature a sustained, still slower, declining phase back to baseline. At the end of each experiment, bradykinin (10 [mu]M) was added to the cells. Bradykinin is known to act through its own G protein-coupled serpentine receptor and to mediate phosphoinositol turnover and IP3-dependent calcium release from intracellular stores (24 ). The bradykinin response, therefore, served as a positive control in each experimental cell for both viability and a responsive calcium signaling pathway. It, further, served as a check on the calibration of the imaging apparatus itself. Note that not every cell responded to bFGF (Fig. 1 G and H), even though its bradykinin response was intact.
To define further the mechanism of bFGF-induced calcium signals in human diploid fibroblasts, and in particular to assess the effect of FGFR3 on calcium signals, we examined the bFGF response of cells with defined mutations in FGFR3. The cells studied, in addition to wild-type, carried defined mutant alleles which produced the distinctive disease phenotypes of homozygous and heterozygous Ach and TD types 1 and 2 (20 ,21 ). Figure 2 presents the analysis of eight representative cells having genotypes causing each of these four disease phenotypes. The studies are carried out in normal extracellular calcium, and are directly comparable with the study of wild-type control cells presented in Figure 1 . These results show that the baseline calcium concentration is indistinguishable for all four disease phenotypes, and that it is the same as found in wild-type control cells, ~100-200 nM. Furthermore, none of the cells show a constitutive signaling phenotype. None of these serum-deprived mutant cells show constitutive calcium waves or even an elevated cytosolic calcium concentration, as would be expected if their FGFRs were constitutively active in the absence of ligand.
In these experiments, we show that in human diploid fibroblasts, as in previously studied model systems, calcium signaling is a useful downstream reporter of physiological FGF receptor activation, and demonstrate that this signal is predominated by a transmembrane calcium influx. We further show that we can use this physiological reporter function to assign a signaling phenotype to single cells, and that the phenotype is highly correlated with certain FGFR3 disease phenotypes.
A coherent interpretation of our results requires us to posit that the mutant FGFR3 alleles function to disrupt signaling in a dominant-negative manner. The results are inconsistent with a constitutive functional activation of signaling by the mutant receptors since the mutant and wild-type cells have the same stable, low basal intracellular calcium concentration during serum starvation, and achieve comparable calcium waves of comparable magnitude and duration during control bradykinin or ATP (not shown) activations. This is also consistent with the well-established observation that these alleles lack any oncogenic potential in vivo or in vitro (1 ,9 ). A dominant-negative interaction, on the other hand, is implied since cells of specific FGFR3 mutant genotypes faithfully remain at basal calcium concentrations after supramaximal bFGF stimulation, a result that could not be achieved without the mutant allele acting through some novel gained function to silence all receptor responses to bFGF. The means by which the mutant receptors achieve negative dominance remains to be determined but, based upon prior studies of engineered dominant-negative FGFR alleles in cells and embryos (12 ,13 ), they must disturb signaling by all expressed allelic and non-allelic FGFRs, most likely through the formation of signaling-incompetent, ligand-dependent or -independent heteromultimers in the plasma membrane.
Genotyped human fibroblast cell cultures were generously provided by Dr J. J. Wasmuth (UC Irvine). Cells were maintained in tissue culture flasks in Dulbecco's minimum essential medium (DMEM) containing 10% fetal bovine serum at 37oC in an atmosphere of 95% air and 5% CO2. For each experiment, cells were plated at low density in the same growth medium on custom-made glass coverslip chambers. After 4-5 days of exponential growth, cells were G0-arrested by culturing in serum-free DMEM medium for the final 24 h preceding imaging.
The cells were loaded with calcium indicator dye by performing a 1 h incubation at 37oC in serum-free DMEM containing 4 [mu]M fura-PE3 (Teflabs). After loading, the cells were washed and maintained in standard extracellular Ringer solution composed of (in mM) 160 NaCl, 4 KCl, 2.0 CaCl2, 1 MgCl2, 5 HEPES, and 10 glucose, adjusted to pH 7.2. For ratiometric imaging of intracellular calcium, light from a 75 W xenon arc lamp was passed through a motorized filter wheel containing 340 and 380 nm filters and a field of the dye-loaded cells imaged using a Zeiss IM 35 inverted microscope. Dye fluorescence was captured by a Hamamatsu C2400 SIT camera, and the signal was processed by a video imaging system (ETM Systems). Eight-bit images, averaged over eight frames, were recorded every 8 s for each wavelength. The raw images were first background subtracted and the resulting images were divided to obtain ratios of the fluorescence at 340 nm/380 nm. Regions of the image were defined to correspond to individual cells, and the intracellular calcium (the fluorescence ratio) within each defined region was followed during the course of the experiment. Depending upon the density of the cells in a culture, between 10 and 50 cells could be distinguished simultaneously in the imaged field of view. This generates a matrix of intracellular calcium measurements, reported as fluorescence ratios, for each region (cell) versus time. These values are transferred to a spreadsheet program (Sigmaplot) for analysis and figure generation. For dye calibration, the minimal fluorescence ratio, Rmin, was measured using 1 [mu]M ionomycin in Ringer solution buffered to 10 nM CaCl2 (0.1 mM calcium, 11 mM EGTA) and the maximal fluorescence ratio, Rmax, was similarly measured with Ringer solution supplemented to 10 mM CaCl2. Conversion of the fluorescence ratio to a Ca2+ concentration was performed according to Grynkiewicz et al. (38 ).
All experiments were performed at room temperature. The solution in the chamber could be replaced using a perfusion system that takes ~15 s for a 5-fold volume exchange. The bFGF (R & D Systems), bradykinin (Calbiochem) and ATP (Sigma) were solubilized in water as 1000* stocks and stored as single-use aliquots at -30oC. These reagents were diluted into Ringer solution at their final concentrations on the day of experiment.
Basic statistical analysis of means and standard deviations were calculated using either Excel or Sigmastat. Comparisons of datasets by ANOVA were performed using Sigmastat.
We would like to thank Dr John J. Wasmuth, University of California, Irvine, for generously providing the cell lines used in this study and for his continued encouragement. We would like to dedicate this work to the memory of this dynamic and caring pioneer in human genetics. Imaging studies were facilitated through access to the Optical Biology shared resource of the Clinical Cancer Center at University of California, Irvine, a shared resource supplemented by the National Cancer Institute Support Grant CA-62203.
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*To whom correspondence should be addressed. Tel: +1 714 824 7702; Fax: +1 714 824 8540; Email: jjgargus@uci.edu
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