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. 2014 Jun;28(6):2525-37.
doi: 10.1096/fj.13-245936. Epub 2014 Feb 27.

Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations

Holly A Leddy  1 , Amy L McNulty  1 , Suk Hee Lee  2 , Nicole E Rothfusz  1 , Bernd Gloss  3 , Margaret L Kirby  4 , Mary R Hutson  4 , Daniel H Cohn  5 , Farshid Guilak  6 , Wolfgang Liedtke  7
Affiliations

Follistatin in chondrocytes: the link between TRPV4 channelopathies and skeletal malformations

Holly A Leddy et al. FASEB J. 2014 Jun.

Abstract

Point mutations in the calcium-permeable TRPV4 ion channel have been identified as the cause of autosomal-dominant human motor neuropathies, arthropathies, and skeletal malformations of varying severity. The objective of this study was to determine the mechanism by which TRPV4 channelopathy mutations cause skeletal dysplasia. The human TRPV4(V620I) channelopathy mutation was transfected into primary porcine chondrocytes and caused significant (2.6-fold) up-regulation of follistatin (FST) expression levels. Pore altering mutations that prevent calcium influx through the channel prevented significant FST up-regulation (1.1-fold). We generated a mouse model of the TRPV4(V620I) mutation, and found significant skeletal deformities (e.g., shortening of tibiae and digits, similar to the human disease brachyolmia) and increases in Fst/TRPV4 mRNA levels (2.8-fold). FST was significantly up-regulated in primary chondrocytes transfected with 3 different dysplasia-causing TRPV4 mutations (2- to 2.3-fold), but was not affected by an arthropathy mutation (1.1-fold). Furthermore, FST-loaded microbeads decreased bone ossification in developing chick femora (6%) and tibiae (11%). FST gene and protein levels were also increased 4-fold in human chondrocytes from an individual natively expressing the TRPV4(T89I) mutation. Taken together, these data strongly support that up-regulation of FST in chondrocytes by skeletal dysplasia-inducing TRPV4 mutations contributes to disease pathogenesis.

Keywords: bone morphogenetic protein; calcium signaling; cartilage; growth plate.

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Figures

Figure 1.
Figure 1.
Ca2 signaling is increased and FST is up-regulated in huTRPV4V620I porcine chondrocytes. A) Representative Ca2+ traces (each trace is an individual cell) showing chondrocytes with huTRPV4V620I (V620I) or huTRPV4WT (huWT) channels responding to −100 mOsm stimulus. B) Basal Ca2+ levels of chondrocytes with directed expression of V620I or huWT (means±sem; n=19). *P = 0.003; t test on log-transformed data. C) Peak Ca2+ levels of chondrocytes with directed expression of V620I or huWT in response to a −100 mOsm change (means±sem; n=10). *P = 0.0002; t test on log-transformed data. D) Fold change in FST mRNA of chondrocytes expressing huWT and V620I (means±sem; n=7). V620I showed >2-fold up-regulation vs. huWT. E) Western blot showing FST and actin protein levels for chondrocytes with directed expression of V620I or huWT.
Figure 2.
Figure 2.
FST up-regulation requires a TRPV4 channelopathy mutation with a Ca2+-permeable pore and requires the CRE in the FST promoter. A) Fold change in FST mRNA abundance of chondrocytes with directed expression of huTRPV4V620I (V620I) or huTRPV4WT (huWT) with pore blocking (M680K) or monovalent cation permeable but Ca2+ impermeable (M680D) mutations (means±sem; huWT, n=6; V620I, n=6; V620I/M680K, n=4; M680K, n=2; M680D, n=3; V620I/M680D, n=3). *P = 0.0003 vs. all other groups; ANOVA on log-transformed data. B) Western blot showing FST and actin levels for chondrocytes with directed expression of V620I, V620I/M680D, or huWT. Relative luciferase activity of ATDC5 cells that were either undifferentiated (left panel) or chondrogenically differentiated for 3 d (right panel) expressing V620I or huWT, coexpressing a luciferase reporter gene driven by the porcine FST promoter (WT) or the FST promoter containing a mutation in the CRE (mtCRE). Only in differentiated cells was luciferase activity significantly increased in the presence of the V620I mutation as compared to huWT. This increase was completely eliminated when the FST promoter was mutated (means±sem; n=3/group). *P < 0.003; ANOVA.
Figure 3.
Figure 3.
Humanized huTRPV4V620I mice exhibited skeletal changes, increased Ca2+ signaling, and increased Fst expression. A) Length of the femur, tibia, third lower extremity digit (metatarsal plus phalanges of the third digit), third upper extremity digit (metacarpal plus phalanges of the third digit), and L6 vertebrae, as well as the width of the L6 vertebrae (including the transverse process) of the huTRPV4WT (huWT; darker bars) and huTRPV4V620I mice (V620I; lighter bars) (means±sem; n=8). *P < 0.05; t test. B) Faxitron images of the vertebrae. C) Width of the acetabulum, femoral head, and tibial plateau of the huWT and V620I mice (means±sem; n=8). *P < 0.05; t test. D) Percentage of cells in intact femoral cartilage responding with a Ca2+ signal to vehicle control or 10 μM 4αPDD differs between huWT and V620I mice. Bars with different letters differ significantly from one another (P<0.009, χ2; huWT control, n=118; huWT 4αPDD, n=147; V620I control, n=116; V620I 4αPDD, n=119; cells from 3 mice/group). E) Fst mRNA, which was isolated directly from mouse cartilage, was normalized for TRPV4 mRNA in V620I vs. huWT mice. Fst mRNA levels prior to normalization for TRPV4 mRNA were 1.07 ± 0.21 for huWT and 1.38 ± 0.02 for V620I mice (P=0.21; means±sem; n=3). *P = 0.009; t test.
Figure 4.
Figure 4.
Basal and peak Ca2+ levels varied with different TRPV4 mutations, but FST is up-regulated in a TRPV4-dependent manner by humanized porcine chondrocytes expressing skeletal dysplasia causing mutations. A) Basal Ca2+ levels in porcine chondrocytes expressing TRPV4 mutations (means±sem; huWT, n=38; V620I, n=50; A716S, n=36; T89I, n=16; F273L, n=20). *P < 0.00001 vs. huWT; ANOVA on log-transformed data. B) Peak Ca2+ levels in response to −100 mOsm change in porcine chondrocytes expressing TRPV4 mutations (means±sem; huWT, n=28; V620I, n=40; A716S, n=26; T89I, n=6; F273L, n=15). *P < 0.0001 vs. huWT; ANOVA on log-transformed data. C) Fold change of FST mRNA with different TRPV4 mutations without (darker bars) and with (lighter bars) selective TRPV4 antagonist, GSK205 (25 μM) (means±sem; huWT, n=10; V620I, n=9; A716S, n=7; T89I, n=4; F273L, n=3). All dysplasia mutations showed >2-fold up-regulation. D) Fold change of FST mRNA abundance for huWT, A716S, and A716S/M680D (means±sem; n=3). *P = 0.003 vs. other groups; ANOVA.
Figure 5.
Figure 5.
FST decreases ossification of developing chick limbs. A) Representative images of chick tibia stained with alizarin red (bone) and alcian blue (cartilage). Red circles indicate location of PBS- or FST-soaked beads near the tibia of the right legs. Scale bar = 500 μm. B) Percentage ossification of untreated (darker bars) and treated (with a PBS- or FST-soaked bead; lighter bars) tibiae (means±sem; PBS, n=14; FST, n=18). *P = 0.01 vs untreated bead; paired t test. C) Percentage ossification of untreated (darker bars) and treated (with a PBS- or FST-soaked bead; lighter bars) femurs. means±sem; PBS, n=14; FST, n=18). *P = 0.02 vs. untreated bead; paired t test.
Figure 6.
Figure 6.
Case study of primary human chondrocytes from a TRPV4T89I individual demonstrates increased FST expression; model to explain how TRPV4 mutations may cause skeletal dysplasia. A) Relative FST mRNA levels corrected for relative TRPV4 mRNA levels in human control chondrocytes and chondrocytes from a lethal TRPV4T89I (T89I) case. Prior to normalization for TRPV4 mRNA, FST mRNA levels were 2.4-fold higher in the T89I chondrocytes (5.79), as compared to control chondrocytes (2.41±0.69). Bars represent means ± sem. B) FST protein secreted by control chondrocytes or chondrocytes from a patient with the TRPV4 T89I mutation. Data are expressed as picograms FST per microgram RNA. Control, n = 7; T89I, n = 1. Bars represent means ± sem. C) Our proposed model of how TRPV4 mutations cause skeletal dysplasia. The skeletal dysplasia mutant TRPV4 channels allow altered Ca2+ influx into chondrocytes. Ca2+ signaling facilitates phosphorylation and binding of CREB to the CRE in the FST promoter (45, 46), thus up-regulating FST expression. In turn, FST functions as a BMP inhibitor in the extracellular milieu, thus inhibiting BMP activity (25, 26), and this attenuation of BMP signaling prevents proper bone formation, resulting in skeletal dysplasias.
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