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  • The DDRs can interact with multiple proteins and also modula

    2020-07-28

    The DDRs can interact with multiple proteins and also modulate signaling pathways initiated by other matrix receptors, cytokines,growth factors, and transmembrane receptors in a context- and cell-type-dependent manner.3, 4 We therefore evaluated the consequences of DDR2 activation on potential downstream growth-stimulatory pathways and STAT1. The latter is an important modifier in the overgrowth and tissue wasting seen in individuals with PDGFRB [MIM: 173410] gain-of-function variants. The following proteins were assessed with appropriate antibodies, all obtained from Cell Signaling Technology at recommended dilutions (see Supplemental Methods for details): phospho-Tyr542-PTPN11(SHP-2), phospho-Tyr580-PTPN11(SHP-2), PTPN11(SHP-2), phospho-Ser473-AKT, AKT, phospho-Thr202/Tyr204-MAPK3(ERK1), MAPK3(ERK1), phospho-Tyr416-SRC, Tyr416-SRC, phospho-Tyr527-SRC, Tyr527-SRC, SRC, and phospho-Tyr70-STAT1. We did not detect increased phosphorylation of any of these proteins (Figures S7–S21). This suggests that the consequence of DDR2 activation in Warburg-Cinotti syndrome (MIM: 618175) is targeted to a group of proteins with little signal-transduction crosstalk with well-known growth-stimulatory pathways, such as the RAS/ERK and PI3K/AKT pathways. Our finding that activating DDR2 variants are a cause of this disease suggested that the ABL inhibitor dasatinib, a leukemia drug that also inhibits DDR2, could be used for treatment of affected individuals.10, 11 To examine the effect of dasatinib on p.Leu610Pro- and p.Tyr740Cys-induced autophosphorylation, we cultured fibroblasts from affected individuals and controls as described above. When the Aminophylline australia were 80%–90% confluent, the medium was replaced with serum-free DMEM. After 16 hours, cells were either left untreated or treated with 0.05 or 0.1μM dasatinib (#S1021, Selleckchem); they were then harvested after 6 hours as described above. Immunoblot analysis determined the presence of phospho-Tyr740-DDR2. At both concentrations, dasatinib abolished the observed autophosphorylation of DDR2 (Figure4C and Figures S22 and S23), providing invitro support for experimental treatment of affected individuals. Penttinen syndrome, associated with activating mutations in PDGFRB, and Warburg-Cinottisyndrome have many similarities, such as lipodystrophy, subcutaneous-tissue wasting and accompanying hypertrophic lesions, and marked acro-osteolysis. Of note, PDGF-targeted therapy has been effective in three reported individuals with germline activating PDGFRB mutations.13, 14 DDR2 is an important regulator of bone growth and resorption, both as a promoter of osteoblastogenesis and as an inhibitor of osteoclastogenesis.15, 16 DDR2 has been suggested as a therapeutic target for osteoporosis. In addition, DDR2-collagen interaction stimulates the secretion of lysyl oxidase, which cross-links collagen fibers in the ECM.3, 17 Why activating mutations in DDR2 might be associated with osteolysis in the individuals described here remains to be elucidated. However, bi-allelic loss-of-function variants in DDR2 cause spondylometaepiphyseal dysplasia accompanied by short limbs and abnormal (premature) calcifications (SMED-SL [MIM: 271665]).18, 19 This developmental disorder is associated with decreased bone formation but not increased bone destruction (or osteolysis). No skin or eye changes have been reported.18, 19, 20 The individuals described in this report had normal or tall stature and normal limb lengths, indicating normal developmental bone growth. They also had an acquired arthropathy with osteolysis that was associated with flexion contractures. Premature calcifications (as seen in SMED-SL) were not observed in any of the individuals with Warburg-Cinotti syndrome. Thus, the phenotype of the individuals reported here with gain-of-function DDR2 variants was distinct from that seen in SMED-SL, which is associated with loss-of-function DDR2 variants.