PDE3A mutations cause autosomal dominant hypertension with brachydactyly (original) (raw)

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References

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Acknowledgements

We thank all family members for their cooperation. We thank M.-B. Köhler and M. Toliat for technical assistance. P.G.M., F.C.L., O.T. and S.B. received support from the Deutsche Forschungsgemeinschaft (DFG; BA1773/4-1, BA1773/4-2, MA5028/1-2 and MA5028/1-3) and grants-in-aid from the German Hypertension Society (Deutsche Hochdruckliga, DHL) and from the German Heart Research Foundation (F/24/13). E.K. was supported by the DFG (KL1415/4-2), the Else Kröner-Fresenius-Stiftung (2013_A145) and the German-Israeli Foundation (I-1210-286.13/2012). F.C.L. received support from the Lingen-Stiftung. F.V. and M.A.M. were supported by the US Department of Veterans Affairs (CARA-029-09F), the American Heart Association (10034439) and the University of Utah Research Foundation. James C. Melby referred one of the families. Dr. Melby died on 19 August 2007.

Author information

Author notes

  1. Philipp G Maass
    Present address: Present address: Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts, USA.,
  2. Philipp G Maass, Atakan Aydin, Friedrich C Luft, Carolin Schächterle and Sylvia Bähring: These authors contributed equally to this work.

Authors and Affiliations

  1. Experimental and Clinical Research Center (ECRC), a joint cooperation between the Charité Medical Faculty and the Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
    Philipp G Maass, Atakan Aydin, Friedrich C Luft, Martin Vaegler, Fatimunnisa Qadri, Knut Mai, Maolian Gong & Sylvia Bähring
  2. Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany
    Philipp G Maass, Atakan Aydin, Friedrich C Luft, Carolin Schächterle, Fatimunnisa Qadri, Herbert Schulz, Irene Hollfinger, Yvette Wefeld-Neuenfeld, Eireen Bartels-Klein, Astrid Mühl, Russell Hodge, Maolian Gong, Franz Rüschendorf, Norbert Hübner, Enno Klussmann & Sylvia Bähring
  3. Division of Clinical Pharmacology, Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA
    Friedrich C Luft
  4. Institute of Human Genetics, Jena University Hospital, Friedrich Schiller University, Jena, Germany
    Anja Weise, Katharina Rittscher & Thomas Liehr
  5. Max Planck Institute for Molecular Genetics, Berlin, Germany
    Sigmar Stricker, Peter M Krawitz, Dmitri Parkhomchuk, Jochen Hecht, Thomas F Wienker & Stefan Mundlos
  6. Institute for Chemistry and Biochemistry, Freie Universität Berlin, Berlin, Germany
    Sigmar Stricker
  7. Department of Nephrology, Hannover University Medical School, Hannover, Germany
    Carsten Lindschau & Hermann Haller
  8. Staatliche Technikerschule Berlin, Berlin, Germany
    Carsten Lindschau
  9. Department of Urology, Laboratory of Tissue Engineering, Eberhard Karls University Tübingen, Tübingen, Germany
    Martin Vaegler
  10. Division of Nephrology and Hypertension, Eastern Virginia Medical School, Norfolk, Virginia, USA
    Hakan R Toka
  11. Division of Nephrology, Brigham and Women's Hospital, Boston, Massachusetts, USA
    Hakan R Toka
  12. Cologne Center for Genomics (CCG), University of Cologne, Cologne, Germany
    Herbert Schulz
  13. Institute for Medical Genetics and Human Genetics, Charité Universitätsmedizin Berlin, Berlin, Germany
    Peter M Krawitz, Dmitri Parkhomchuk & Stefan Mundlos
  14. Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Berlin, Germany
    Peter M Krawitz, Dmitri Parkhomchuk, Jochen Hecht & Stefan Mundlos
  15. Department II of Medicine, University of Cologne, Cologne, Germany
    Martin Kann
  16. Center for Molecular Medicine Cologne, University of Cologne, Cologne, Germany
    Martin Kann
  17. INFOGEN, Berlin, Germany
    Herbert Schuster
  18. Division of Clinical and Metabolic Genetics, The Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada
    David Chitayat
  19. Department of Obstetrics and Gynecology, Prenatal Diagnosis and Medical Genetics Program, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada
    David Chitayat
  20. Division of Medical Genetics, North Shore/LIJ Health System, Manhasset, New York, USA
    Martin G Bialer
  21. Department of Pediatrics, North Shore/LIJ Health System, Manhasset, New York, USA
    Martin G Bialer
  22. Institute for Medical Biometry, Informatics and Epidemiology, University of Bonn, Bonn, Germany
    Thomas F Wienker
  23. Institute of Psychology, Chinese Academy of Sciences, Beijing, China
    Jürg Ott
  24. Statistical Genetics, Rockefeller University, New York, New York, USA
    Jürg Ott
  25. Institute of Clinical Pharmacology, Hannover Medical School, Hannover, Germany
    Jens Jordan & Jens Tank
  26. Centre Hospitalier Universitaire de Caen, Cytogénétique Postnatale et Génétique Clinique, Caen, France
    Ghislaine Plessis
  27. Department of Neurosurgery, Bundeswehrkrankenhaus Ulm, Ulm, Germany
    Ramin Naraghi
  28. Department of Pediatrics, Griffith Base Hospital, Griffith, New South Wales, Australia
    Maxwell Hopp
  29. Department of Ophthalmology, Hospital Ludwigshafen, Ludwigshafen, Germany
    Lars O Hattenbach
  30. HealthTwist, Berlin, Germany
    Andreas Busjahn
  31. Institute for Medical Genetics, University of Zurich, Zurich, Switzerland
    Anita Rauch
  32. Cardiology Section, Veterans Affairs Salt Lake City Health Care System, Salt Lake City, Utah, USA
    Fabrice Vandeput & Matthew A Movsesian
  33. Department of Internal Medicine, University of Utah, Salt Lake City, Utah, USA
    Fabrice Vandeput & Matthew A Movsesian
  34. Department of Pharmacology and Toxicology, University of Utah, Salt Lake City, Utah, USA
    Fabrice Vandeput & Matthew A Movsesian
  35. DZHK (German Centre for Cardiovascular Research), Berlin, Germany
    Norbert Hübner & Enno Klussmann
  36. Charité Universitätsmedizin, Berlin, Germany
    Norbert Hübner
  37. Department of Pediatric Oncology, Hacettepe University, Ankara, Turkey
    Nihat Bilginturan
  38. Department of Pediatric Cardiology, Children's Hospital, Friedrich Alexander University Erlangen, Erlangen, Germany
    Okan Toka

Authors

  1. Philipp G Maass
  2. Atakan Aydin
  3. Friedrich C Luft
  4. Carolin Schächterle
  5. Anja Weise
  6. Sigmar Stricker
  7. Carsten Lindschau
  8. Martin Vaegler
  9. Fatimunnisa Qadri
  10. Hakan R Toka
  11. Herbert Schulz
  12. Peter M Krawitz
  13. Dmitri Parkhomchuk
  14. Jochen Hecht
  15. Irene Hollfinger
  16. Yvette Wefeld-Neuenfeld
  17. Eireen Bartels-Klein
  18. Astrid Mühl
  19. Martin Kann
  20. Herbert Schuster
  21. David Chitayat
  22. Martin G Bialer
  23. Thomas F Wienker
  24. Jürg Ott
  25. Katharina Rittscher
  26. Thomas Liehr
  27. Jens Jordan
  28. Ghislaine Plessis
  29. Jens Tank
  30. Knut Mai
  31. Ramin Naraghi
  32. Russell Hodge
  33. Maxwell Hopp
  34. Lars O Hattenbach
  35. Andreas Busjahn
  36. Anita Rauch
  37. Fabrice Vandeput
  38. Maolian Gong
  39. Franz Rüschendorf
  40. Norbert Hübner
  41. Hermann Haller
  42. Stefan Mundlos
  43. Nihat Bilginturan
  44. Matthew A Movsesian
  45. Enno Klussmann
  46. Okan Toka
  47. Sylvia Bähring

Contributions

N.B. first described this syndrome in 1973. F.C.L. and his laboratory have pursued this project since 1994. O.T., H.R.T., H. Schuster, J.J., J.T., H.H., R.H., L.O.H. and R.N. phenotyped the syndrome. D.C., M.G.B., G.P., M.H. and H.R.T. identified additional families with the syndrome. T.F.W., J.O., S.B., A.B. and F.R. performed microsatellite and SNP linkage analyses. M.G. and N.H. performed genotyping analyses within the families and also analyzed Chinese hypertensive families that showed linkage to the chromosome 12p locus. A.W., M.K., A.R., K.R. and T.L. performed cytogenetics. S.S. performed in situ mouse studies. S.M., P.M.K., D.P. and J.H. carried out Illumina whole-genome sequencing. A.A., P.G.M. and S.B. analyzed Complete Genomics whole-genome sequencing data, and A.A. identified the PDE3A mutation. H. Schulz statistically analyzed various data. C.L. and A.A. performed the confocal immunofluorescence imaging. F.Q., I.H., E.B.-K. and A.M. performed technical studies. K.M. and Y.W.-N. prepared MSCs. M.V. kindly provided unaffected MSCs and supported all the MSC investigations. Y.W.-N., A.A. and P.G.M. analyzed cell proliferation. F.V. and M.A.M. provided Flag-tagged PDE3A expression constructs and provided intellectual input. C.S. and E.K. performed ELISA assays on recombinant proteins and peptide SPOT assays. P.G.M. participated in all scientific aspects of the study and was personally responsible for the PDE3A functional assays, IC50 determinations and work with MSCs. P.G.M., F.C.L. and S.B. wrote the manuscript. The manuscript was the product of more than 20 years of research to which all authors have contributed.

Corresponding author

Correspondence toFriedrich C Luft.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PDE3A conservation and whole-genome sequencing.

(a) Multiple-sequence alignment of the PDE3A peptide sequence near the identified mutations in five different species. The altered residues were in a highly conserved PDE3A domain. (b) Genomic coverage of Complete Genomics (CG) whole-genome sequencing of Turkish family members IV/6, IV/7, V/14 and V/30. A mean coverage of 57.39 ± 0.25× for each of the four samples was reached. (c) The detected insertion and deletion (indel) events compared to genome assembly hg19. (d) The substitution events are summarized. To use the complementary advantage of different genome sequencing platforms, we performed whole-genome sequencing of the same Turkish patient, whose DNA was also sequenced by CG, on the Illumina platform to screen for further small sequence variations, structural variants and the inversion breakpoints, which were found in fibroblasts and LCLs by interphase FISH. Among the more than 3 million SNVs and small indels, we identified the missense mutation in PDE3A that was also detected in CG sequencing. c.1334C>A in exon 4 of PDE3A (NM_000921; p.T445N) affects a highly conserved amino acid and was not observed in 1000 Genomes Project data nor among 5,000 exomes (Exome Variant Server, http://evs.gs.washington.edu/EVS/; accessed July 2013), in Sanger sequencing of all non-affected family members or in 200 unrelated Caucasian controls. (e) Electropherograms of the six different missense mutations in PDE3A that introduced amino acid changes at positions 445, 447 and 449; total number of samples analyzed; AFF, affected individuals; NON, unaffected controls.

Supplementary Figure 2 Magnetic resonance imaging (MRI) of VI/9.

(a) MRI of the hypertensive teenager with mild BDE (VI/9). The red arrows indicate the contacting vessels to the ventrolateral medulla (VLM) on the left and right sides. On the left side, the PICA is the offending artery indicating a type 1 neurovascular contact (nvc). The yellow arrows indicate the cranial nerves IX & X. (b) The red arrows indicate the contacting vessels to the VLM. (c) The upper image shows the left VLM. The red arrows indicate the PICA as an impressive loop contacting the VLM as type 1 nvc. The arrows in the middle image document the PICA attached to the right VLM. The coronal projection displays the vessel signal on both sides of the VLM.

Supplementary Figure 3 Pde3a in situ hybridization and characterization of human chondrogenically induced fibroblasts.

(a) Murine in situ hybridization with 3′ UTR probe in Pde3a detected light limb bud expression in embryos of 12.5 and 13.5 d. (b) The in situ antisense control probe in the coding sequence of Pde3a (cds) showed no expression in the developing limb buds. (c) Successful chondrogenic induction of human fibroblasts from buttock biopsies after 3 weeks in pellet culture. We used chondrogenically induced fibroblasts to focus on chondrogenic PTHLH expression. Because we were able to obtain more fibroblasts from non-affected and related controls than MSCs from the Turkish family, we performed PTHLH expression experiments on chondrogenically induced fibroblasts. After embedding and sectioning, Alcian blue or Safranin O staining detected extracellular matrix proteins that are characteristic for cartilage.

Supplementary Figure 4 cAMP and cGMP quantifications, and Lineweaver-Burke plots of Michaelis-Menten kinetics.

(a,b) HeLa cells were transiently transfected with the full-length wild-type PDE3A construct and the six full-length PDE3A mutant constructs; forskolin or l-arginine stimulation was performed to enhance intracellular cAMP or cGMP levels through adenylate or guanylate cyclase activation. Forty-eight hours after transfection, cells were collected to determine cAMP (a) and cGMP (b) levels in enzyme immunoassays. The data shown are the results of three independent experiments with minimum and maximum deviation. Significant differences were determined between wild-type PDE3A (wt) and the mutants, defining the mutations as gain-of-function mutations, causing increased cAMP hydrolysis (n = 4; P ≤ 0.001, two-tailed Student’s t test). cGMP levels were not altered in the presence of the PDE3A mutants (n = 6). (c) Western blotting detected successful expression of wild-type and mutant PDE3A proteins. Controls were cells transfected with empty vector. (d–g) Linear regression analysis of transformed Michaelis-Menten data from Figure 3a of Flag-tagged PDE3A1-WT and PDE3A1-T445N. (h–k) Linear regression analysis of transformed Michaelis-Menten data from Figure 3b of Flag-tagged PDE3A2-WT and PDE3A2-T445N, visualized as Lineweaver-Burke plots.

Supplementary Figure 5 IC50 measurement of cGMP.

cGMP competitively inhibits PDE3A, but nearly equivalent values were determined for Flag-tagged wild-type (WT) PDE3A1 and the tagged PDE3A1-T445N mutant. IC50 was 5.164 µM for WT and 4.78 µM for T445N (n = 3). Relevant differences were not found in three independent experiments.

Supplementary Figure 6 Functional CRE-luciferase assays in HeLa cells transiently expressing the six PDE3A mutants.

(a) HeLa cells were transfected with an empty vector (sc300-w/o; orange line), a full-length wild-type PDE3A expression construct (red line) and the six full-length PDE3A mutant expression plasmids (gray and black lines). HeLa cells were cotransfected with a cAMP-responsive element (CRE) regulating luciferase transcriptional activity under the influence of increasing forskolin concentrations to further elucidate the functional consequences of the PDE3A mutations (P < 0.002). A Renilla luciferase vector was used for standardization. The data describe the relative increase in luciferase activity normalized to the DMSO control of cells transfected with empty vector. The results are the means of three independent experiments (mean ± s.d.; n = 3; Wilcoxon-Mann-Whitney test, P < 0.002). The more hydrolyzed cAMP there was, the less luciferase expression was detected. In the presence of increasing forskolin concentrations enhancing cAMP levels, the PDE3A mutants showed a significant reduction in CRE-mediated luciferase activity as a result of the higher cAMP hydrolysis compared to wild-type PDE3A. (b) cGMP stimulation with increasing l-arginine concentrations showed that cGMP competitively inhibited cAMP hydrolysis with a significant difference between the mutants and wild-type PDE3A (P < 0.002). The more cGMP that was present, the less cAMP hydrolysis occurred and the greater the luciferase activity was.

Supplementary Figure 7 Characterization of MSCs and MSC-derived cells.

(a) FACS results of the affected patient VI/17 and one non-affected control. The analysis of surface markers detected CD105+, CD90+, CD73+, HLA-ABC+, CD31–, CD34–, CD45– and HLA-DR– cells. (b) Plastic adherence and the multilineage potential of MSCs of one non-affected control and VI/17. MSCs and VSMCs from patient VI/9 and the second non-affected control were identically characterized and fulfilled all criteria of MSCs and VSMCs (data not shown). Immunocytochemical staining of the MSC-derived adipocytes, osteocytes and chondrocytes validated the multilineage potential of the MSCs. The fatty vacuoles show successful differentiation into adipocytes; calcium precipitates characterized MSC-derived osteocytes. Toluidine blue stained proteoglycans of sectioned chondrogenic tissue that was generated in micromass pellet cultures. Successful differentiation was similar in the MSCs from the control and affected patient VI/17. MSC characterizations fulfilled the criteria of the International Society for Cellular Therapy. (c) Myogenic differentiation of one non-affected control and MSCs from VI/17 into VSMCs after 21 d of differentiation. Semiquantitative immunofluorescence detected the smooth muscle markers smooth muscle actin (SMAα), calponin and transgelin (SM22α), which were highly expressed compared to in undifferentiated MSCs. The color spectrum indicates the expression level from low (black) to high (red).

Supplementary Figure 8 PDE3A overexpression in HeLa cells and carboxyfluorescein diacetate succinimidyl ester (CFSE) signal quantification in FACS analysis.

Intensity histogram of flow cytometry analysis of CFSE-labeled and transfected HeLa cells. After CFSE labeling, the cells were seeded and were transfected the next day with full-length wild-type PDE3A or full-length PDE3A expressing the six mutations. Measurements were performed at 24, 48 and 72 h after transfection to determine the proliferation rates. Because of mitosis and the distribution of CFSE, the signal intensity changed in the measured time of 72 h.

Supplementary Figure 9 Individual western blotting experiments.

(a–h) Individual quantification of the western blot signals for each PDE3A mutant in the three independent experiments shown in Figure 4a,b. The signals were densitometrically quantified in relation to the loading control β-tubulin. Because of experimental variation and quality differences between the antibodies to phosphorylated protein, we observed variations in the investigated phosphorylation of Ser428 and Ser438 in PDE3A1 and PDE3A2. The antibody to PDE3A (Bethyl) recognizes motifs beginning at position 450. Perhaps the G449V substitution alters the neighboring protein domain and alters antibody binding, leading to the weak signals in the upper blots. However, the pooled results (Fig. 4a,b) of all mutants showed statistical significance compared to wild-type PDE3A (WT) in non-parametrical Mann-Whitney rank-sum testing.

Supplementary Figure 10 PKA and PKC phosphorylate PDE3A Ser438.

(a) Full-length wild-type (WT; NM_000921) and T445N PDE3A were transiently expressed in HeLa cells. All vectors encoded both PDE3A1 and PDE3A2, which arise from the use alternative translational start sites. In forskolin-stimulated cells, T445N PDE3A Ser438 phosphorylation was increased compared to WT and mock control (sc300 w/o). Use of the PKA inhibitor H89 showed less Ser438 phosphorylation. PDE3A-T445N phosphorylation was still stronger at Ser438 compared to controls. VASP Ser157 phosphorylation that is mediated by PKA was reduced in H89 PKA-inhibited cells. Non-phosphorylated VASP was not altered. (b) Use of PKC α, β, δ, ɛ and γ inhibitor bisindolylmaleimide I showed that PDE3A Ser438 was also phosphorylated by PKC. The combination of H89 with bisindolylmaleimide I further reduced Ser438 phosphorylation. Endogenous VASP was apparently not affected. (c) PMA stimulation alone and in combination with bisindolylmaleimide I and/or H89 inhibitors showed decreased PDE3A Ser438 and VASP Ser157 phosphorylation. Detection of non-phosphorylated VASP showed no obvious differences. However, the data suggest that other protein kinases also phosphorylate Ser438. Tubulin was used as a loading control; representative blots are shown for n = 3 experiments.

Supplementary Figure 11 Pyrosequencing of the PDE3A mutation encoding T445N in MSCs from VI/17.

The figure shows the raw data from PDE3A pyrosequencing of the patient harboring the T445N substitution. The data shown are from a reverse pyrosequencing approach. The yellow area indicates the position of the PDE3A mutation. The base “G” varies between 50 and 100%, and the base “T” varies between 0 and 50%. The data shown are from three independent replicates; error bars, s.d. The presence of each allele in the reaction is displayed as a percentage. Compared to the wild-type allele “G” (G = 99%, T = 1%), no significant differential expression was detected for the mutated PDE3A “T” allele of the patient harboring the T445N substitution (G = 55%, T = 45%). Preferred monoallelic expression, due to either the mutation or the inversion, was excluded.

Supplementary Figure 12 Expression of PTHrP and VASP in MSCs and VSMCs.

The color spectrum indicates expression levels from low (black) to high (red) in semiquantitative immunofluorescence. (a) PTHrP influences the proliferation of VSMCs (Song, G.J., Fiaschi-Taesch, N. & Bisello, A. Mol. Endocrinol.23, 1681-1680, 2009). Full-length PTHrP protein levels were increased, whereas PTHrP peptide levels were decreased, in MSCs and VSMCs from the patient harboring the T445N substitution. (b) Phosphorylation of VASP at Ser157 was reduced in VSMCs from the affected patients compared to controls; phosphorylated VASP was not detectable in MSCs.

Supplementary Figure 13 Peptide assays for the PDE3A mutants.

(a) Amino acid sequences of the synthesized peptide spots from Figure 5a. Serine residues (S; red) at position 428 or 438 were replaced by alanine (A; blue) or aspartic acid (D; green) or with prephosphorylated serine (pS; pink). Positions 445, 447 and 449 for the altered amino acids are marked in yellow; the altered amino acid is marked in gray. (b) Phosphorylation of two peptide spot membranes (30-mers representing PDE3A Ile421-Leu450; see Fig. 2a) without (– PKA) and with (+ PKA) PKA. The peptide spots for PDE3A-WT and PDE3A-T445N (black frame) are the same signals, shown in Figure 5a. (c) Amino acid sequences of the synthesized peptide spots from b. The color code is as indicated above. Serine residues at position 428 or 438 were replaced by alanine or aspartic acid or by prephosphorylated serine. Positions 445, 447 and 449 corresponding to the altered amino acids are shown in yellow; each altered amino acid is marked in gray.

Supplementary Figure 14 Individual measurements of peptide spot quantifications and further peptide sequences.

(a–c) The individual signal quantifications for the peptide spot experiments shown as pooled results in Figure 5b. The summarized data for all mutations from lanes 6, 7 and 9 were statistically significant, as determined by non-parametrical two-tailed Mann-Whitney rank-sum testing (Fig. 5b). (d) Signal quantifications for the six PDE3A mutant peptides and wild-type PDE3A (WT) of lane 3 from Figure 5a and Supplementary Figure 13b (Ser428 and Ala438) showed no significant changes after PKA phosphorylation, indicating that Ser428 is not differentially phosphorylated by PKA. (e) Peptide assay to further determine the signal intensities at prephosphorylated Ser438 with alanine (A; blue) or aspartic acid (D; green) replacements or prephosphorylated serine (pS; pink) for the three different serine residues (S; red) at positions 428, 438 and 439 and the threonine residue (T; dark red) at position 440. (f) Amino acid sequences of the synthesized peptide spots from e. The serine residue at position 428 or 438 was replaced by alanine or aspartic acid or by prephosphorylated serine. The color code is as indicated above.

Supplementary Figure 15 PDE3A and its signal transductions.

Scheme for the involvement of PDE3A in VSMCs and chondrocytes. In VSMCs, the myosin light chain kinase (MLCK) and the myosin light chain phosphatase (MLCP) act in concert to phosphorylate and dephosphorylate the myosin light chain (MLC) for vascular contraction and relaxation, respectively (Pfitzer, G. J. Appl. Physiol 91, 497-503, 2001). PKA and protein kinase G (PKG) activate VASP by phosphorylation of Ser157 and Ser239, respectively. Reduced VASP Ser157 phosphorylation and higher PDE3A activity were associated with enhanced VSMC proliferation that accounts for vessel wall hyperplasia (Zhao, H., Guan, Q., Smith, C.J. & Quilley, J. et al. Eur. J. Pharmacol. 590, 29-35, 2008). Full-length PTHrP stimulated whereas PTHrP peptide (1–36) repressed VSMC proliferation (Song, G.J., Fiaschi-Taesch, N. & Bisello, A. Mol. Endocrinol. 23, 1681-1680, 2009). CREB binding to the PTHLH promoter regulates PTHLH expression in a cAMP-dependent manner. The encoded protein PTHrP transduces signals through the PTH1R (PTH/PTHrP) receptor in chondrocytes and activates the heterotrimeric G protein Gs, thereby stimulating adenylate cyclase (AC) to produce cAMP (Bastepe, M. et al. Proc. Natl. Acad. Sci. USA 101, 14794–14799, 2004). Because PDE3A hydrolyses cAMP, PKA is regulated in a cAMP-dependent manner and activates CREB, which in turn regulates PTHLH expression (Chilco, P.J., Leopold, V. & Zajac, J.D. Mol. Cell. Endocrinol.138, 173–184, 1998).

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Maass, P., Aydin, A., Luft, F. et al. PDE3A mutations cause autosomal dominant hypertension with brachydactyly.Nat Genet 47, 647–653 (2015). https://doi.org/10.1038/ng.3302

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