title: Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on osteoporotic bone.
abstract: Withania somnifera or Ashwagandha is a medicinal herb of Ayurveda. Though the extract and purified molecules, withanolides, from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been studied. Here, we show that one of the withanolide, withaferin A (WFA) acts as a proteasomal inhibitor (PI) and binds to specific catalytic β subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted osteoblast survival and suppressed inflammatory cytokines. In osteoclast, WFA treatment decreased osteoclast number directly by decreasing expression of tartarate-resistant acid phosphatase and receptor activator of nuclear factor kappa-B (RANK) and indirectly by decreasing osteoprotegrin/RANK ligand ratio. Our data show that in vitro treatment of WFA to calvarial osteoblast cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic protein 2. Increased Smurf2 expression due to exogenous treatment of tumor necrosis factor α (TNFα) to primary osteoblast cells was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral administration of WFA to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur, decreased bone turnover markers (osteocalcin and TNFα) and expression of skeletal osteoclastogenic genes. It also increased new bone formation and expression of osteogenic genes in the femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid hormone and alendronate (FDA approved drugs). WFA promoted the process of cortical bone regeneration at drill-holes site in the femur mid-diaphysis region and cortical gap was bridged with woven bone within 11 days of both estrogen sufficient and deficient (ovariectomized, Ovx) mice. Together our data suggest that WFA stimulates bone formation by abrogating proteasomal machinery and provides knowledge base for its clinical evaluation as a bone anabolic agent.
authors: Division of Endocrinology and Center for Research in Anabolic Skeletal Targets in Health and Illness (ASTHI), CSIR-Central Drug Research Institute, Lucknow 226001, India.
authors: V
authors: V
authors: Khedgikar
authors: P
authors: P
authors: Kushwaha
authors: J
authors: J
authors: Gautam
authors: A
authors: A
authors: Verma
authors: B
authors: B
authors: Changkija
authors: A
authors: A
authors: Kumar
authors: S
authors: S
authors: Sharma
authors: G K
authors: GK
authors: Nagar
authors: D
authors: D
authors: Singh
authors: P K
authors: PK
authors: Trivedi
authors: N S
authors: NS
authors: Sangwan
authors: P R
authors: PR
authors: Mishra
authors: R
authors: R
authors: Trivedi
mesh_terms: D045930:Anabolic Agents / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D001696:Biomechanical Phenomena / Q000187:drug effects; D001861:Bone Regeneration / Q000187:drug effects / Q000235:genetics; D001842:Bone and Bones / Q000187:drug effects / Q000473:pathology* / Q000503:physiopathology; D002113:Calcification, Physiologic / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D050659:Core Binding Factor Alpha 1 Subunit / Q000378:metabolism; D005260:Female; D005269:Femur / Q000187:drug effects / Q000473:pathology / Q000503:physiopathology; D051379:Mice; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010010:Osteoclasts / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D010012:Osteogenesis / Q000187:drug effects / Q000235:genetics; D010024:Osteoporosis / Q000188:drug therapy* / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D010052:Ovariectomy; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology / Q000627:therapeutic use; D059748:Proteolysis / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054358:Withanolides / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology* / Q000627:therapeutic use; D014945:Wound Healing / Q000187:drug effects / Q000235:genetics
chemical_list: D045930:Anabolic Agents; D015415:Biomarkers; D050659:Core Binding Factor Alpha 1 Subunit; D061988:Proteasome Inhibitors; C493716:Runx2 protein, mouse; D014409:Tumor Necrosis Factor-alpha; D054358:Withanolides; C533325:Smurf2 protein, mouse; D044767:Ubiquitin-Protein Ligases; D046988:Proteasome Endopeptidase Complex; C009684:withaferin A
references: Metabolism. 2002 Sep;51(9):1153-60
references: 12200760
references: Eur J Pharm Biopharm. 2012 Nov;82(3):508-17
references: 22926146
references: Science. 2000 Sep 1;289(5484):1504-8
references: 10968780
references: J Clin Invest. 2003 Jun;111(11):1771-82
references: 12782679
references: Nat Clin Pract Rheumatol. 2006 Jan;2(1):35-43
references: 16932650
references: Osteoporos Int. 2001;12(5):385-90
references: 11444087
references: PLoS One. 2013 May 08;8(5):e62714
references: 23667511
references: Mol Cell Endocrinol. 2010 Aug 30;325(1-2):101-9
references: 20570709
references: Bioorg Med Chem. 2001 Jun;9(6):1499-507
references: 11408168
references: Expert Opin Investig Drugs. 2010 Aug;19(8):995-1005
references: 20629616
references: Phytomedicine. 2012 Sep 15;19(12):1134-42
references: 22951391
references: N Engl J Med. 2003 Jul 24;349(4):327-34
references: 12878739
references: Comput Methods Biomech Biomed Engin. 1997;1(1):15-23
references: 11264794
references: J Biol Chem. 2010 Nov 5;285(45):34828-38
references: 20817730
references: J Orthop Res. 2004 Nov;22(6):1188-92
references: 15475196
references: Mol Pharmacol. 2007 Feb;71(2):426-37
references: 17093135
references: J Orthop Surg Res. 2011 Jun 15;6:30
references: 21676245
references: Mol Cell Biol. 2000 Dec;20(23):8783-92
references: 11073979
references: Bone. 2010 Dec;47(6):1006-12
references: 20807599
references: Int J Mol Med. 2010 Oct;26(4):611-8
references: 20818503
references: Mol Cell Endocrinol. 2009 Apr 10;302(1):86-91
references: 19356626
references: J Med Chem. 2013 Jan 10;56(1):109-22
references: 23214410
references: Growth Factors. 2004 Dec;22(4):233-41
references: 15621726
references: Proc Natl Acad Sci U S A. 1990 Mar;87(6):2172-6
references: 2315310
references: Biochem Biophys Res Commun. 2011 Apr 1;407(1):225-9
references: 21377449
references: Nanomedicine (Lond). 2013 May;8(5):757-71
references: 23311987
references: Gene. 2005 Aug 29;357(1):1-8
references: 16125875
references: Osteoporos Int. 2011 Dec;22(12):3013-27
references: 21225417
references: J Clin Invest. 2006 May;116(5):1186-94
references: 16670759
references: Curr Mol Med. 2010 Feb;10(1):14-28
references: 20205677
references: J Inflamm (Lond). 2009 May 13;6:15
references: 19439083
references: PLoS One. 2007 Oct 24;2(10):e1058
references: 17957242
references: J Biol Chem. 2006 Jun 16;281(24):16502-11
references: 16613856
references: Cell Mol Life Sci. 2013 Jul;70(13):2305-17
references: 23007848
references: J Biol Chem. 2006 Feb 17;281(7):4326-33
references: 16373342
references: BMC Genomics. 2010 Dec 02;11 Suppl 4:S15
references: 21143798
references: J Cell Physiol. 2012 Apr;227(4):1326-34
references: 21604269
references: Proc Natl Acad Sci U S A. 2001 Jan 30;98(3):974-9
references: 11158580
references: In Vivo. 2000 Jan-Feb;14(1):265-8
references: 10757085
references: J Biol Chem. 2002 Jan 25;277(4):2695-701
references: 11723115
references: Menopause. 2012 Dec;19(12):1336-46
references: 22850441
references: Endocr Rev. 2003 Apr;24(2):218-35
references: 12700180
references: Arthritis Res Ther. 2007;9(2):102
references: 17349065
references: J Biol Chem. 2011 Oct 21;286(42):37023-31
references: 21862583
references: Cell Death Dis. 2013 Jan 17;4:e463
references: 23328670
references: J Bone Miner Res. 2011 Sep;26(9):2096-111
references: 21638315
references: Proteomics. 2010 May;10(9):1730-9
references: 20162559
references: Oral Dis. 2002 May;8(3):147-59
references: 12108759
references: Bone. 2005 Mar;36(3):502-11
references: 15777685
references: Cold Spring Harb Perspect Biol. 2010 Mar;2(3):a003350
references: 20300215
references: J Immunol. 1992 Feb 1;148(3):788-94
references: 1309841
references: N Engl J Med. 2003 Oct 23;349(17):1671; author reply 1671
references: 14573741
references: Bone. 2011 Jun 1;48(6):1388-400
references: 21421090
references: J Cell Biochem. 2008 Apr 15;103(6):1707-17
references: 18231993
references: Int J Cancer. 2010 Jul 1;127(1):67-76
references: 19894220
references: J Biol Chem. 2002 Jun 14;277(24):21971-82
references: 11923298
references: Angiogenesis. 2004;7(2):115-22
references: 15516832
references: Bioorg Med Chem Lett. 2012 Jan 15;22(2):890-7
references: 22212722
references: Blood. 2007 Jul 1;110(1):334-8
references: 17371942