Background
Normal development and tissue repair are controlled in part by SMADs, a family of intracellular proteins that are activated by signaling via serine/threonine kinase receptors of the TGF-β superfamily.1 LDN-193189 inhibits SMAD1/5/8 phosphorylation by the bone morphogenetic protein (BMP) type I receptors, which are known as activin receptor-like kinases (ALKs), with an IC50 value of 4.9 nM.2 In in vitro kinase assays, it shows specificity for ALK1, 2, 3, and 6 (IC50s = 0.8, 0.8, 5.3, and 16.7 nM, respectively) over ALK4 and 5 (IC50s = 101 and 350 nM, respectively).3 LDN-193189 has been used to inhibit BMP type I receptor activity to study the pathogenesis of fibrodysplasia ossificans progressive, a congenital hyperossification disorder, and to examine the role of osteogenesis in prostate tumor metastases in bone.4,5
1.Brivanlou, A.H., and Darnell, J.E., Jr.Signal transduction and the control of gene expressionScience295(5556)813-818(2002)
2.Cuny, G.D., Yu, P.B., Laha, J.K., et al.Structure-activity relationship study of bone morphogenetic protein (BMP) signaling inhibitorsBioorg. Med. Chem. Lett.18(15)4388-4392(2008)
3.Sanvitale, C.E., Kerr, G., Chaikuad, A., et al.A new class of small molecule inhibitor of BMP signalingPLoS One8(4)62721(2013)
4.Yu, P.B., Deng, D.Y., Lai, C.S., et al.BMP type I receptor inhibition reduces heterotopic ossificationNat. Med.14(12)1363-1369(2008)
5.Lee, Y.C., Cheng, C.J., Bilen, M.A., et al.BMP4 promotes prostate tumor growth in bone through osteogenesisCancer Res.71(15)5194-5203(2011)
Protocol
Cell experiment: | Mouse PASMCs grown are transiently transfected to 50% confluence in six-well plates with 0.3 μg Id1promoter luciferase reporter construct (BRE-Luc) in combination with 0.6 μg of plasmid expressing constitutively active forms of BMP type I receptors (caALK2, caALK3 or caALK6). For both reporter plasmids, 0.2 μg of pRL-TKRenilla luciferase are used to control for transfection efficiency. PASMCs are incubated with LDN193189 (2 nM-32 μM) or vehicle starting 1 h after transfection. Cell extracts are harvested and quantified relative promoter activity by the ratio of firefly to Renilla luciferase activity with the dual luciferase assay kit[1]. |
Animal experiment: | Mice[2] In the first experiment, SCID mice are implanted with MDA-PCa-118b tumors. After 7 days when tumors reached measurable sizes, mice are injected with LDN193189 (3 mg/kg) or with vehicle intraperitoneally twice a day. Tumor sizes and body weights are measured weekly. Mice are injected with calcein at three days and one day prior to sacrifice. Blood is collected and tumors are weighed. A portion of the tumors are fixed in formaldehyde for micro-computed tomography (microCT), using EVS CT, or further decalcified for bone histomorphometric analysis, using OsteoMeasure Analysis System, or flash frozen for RNA preparation. Osteocalcin in the mouse serum is determined by ELISA. In the second experiment, PCa-118b tumors are first digested with Accumax, and the isolated cells are plated overnight, resuspended in Matrigel in 1:1 ratio, and injected into SCID mice (1×106 cells/mouse) subcutaneously. Mice are treated with LDN193189 five days post-injection. Rats[3] Male Sprague-Dawley (SD) rats, 8 weeks of age, weighing 200-220 g, are purchased from Nanjing Medical University animal center. Rats are randomly assigned to one of seven experiment groups (n=6 per group). Rats are housed with free access to food and water under a natural 12/12 h day/night cycle. The Monocrotaline is administered (60 mg/kg) to rats by subcutaneous injection into the back region. The animal’s lungs are harvested at 28th day of the study after hemodynamic assessment. The UK-92480 group received daily intragastric administration of UK-92480 after the administration of MCT (60 mg/kg). The LDN193189 group received daily intragastric administration of UK-92480 (50 mg/kg) and intraperitoneal injection of LDN193189 (10 mg/kg). In other groups, the same volume saline is given. |
参考文献: [1]. Yu PB, et al. BMP type I receptor inhibition reduces heterotopic [corrected] ossification. Nat Med, 2008, 14(12), 1363-1369.
[2]. Lee YC, et al. BMP4 promotes prostate tumor growth in bone through osteogenesis. Cancer Res, 2011, 71(15), 5194-5203.
[3]. Yang L, et al. UK-92480 increases connexin 40 in smooth muscle cells through activation of BMP pathways in pulmonary arterial hypertension. Int J Clin Exp Pathol. 2014 Jul 15;7(8):4674-84. |
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