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Presentation On Wnt 4 and rhe role of HDAC4 & SIRT1 in bone biology

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Gayathri Vijayakumar
Gayathri Vijayakumar

Wnt proteins are a family of conserved signaling proteins that regulate various processes including cell proliferation, fate determination, and embryonic development. They act through both canonical and non-canonical pathways. Wnt4 specifically promotes female development and suppresses male development. It also contributes to kidney and adrenal cortex development. Studies show Wnt4 enhances stem cell osteogenic differentiation by activating non-canonical pathways like P38MAPK. The NASA-Orthofix project examines the effects of pulsed electromagnetic fields on osteoblast proliferation, differentiation and mineralization using rat primary osteoblasts. Genes analyzed include osteocalcin, BAP, collagen I and GAPDH. Results show PEMF stimulation increases cell count, differentiation markers and mineralization

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GAYATHRI VIJAYAKUMARDr. Nicola C. PartridgeDept of Basic Science & Craniofacial BiologyNYU-CD
WNT PROTEIN FAMILY:Wnt proteins are a family of 19highly conserved cysteine-rich secreted glyco-lipoproteins (39-46 Kda) that regulate development, cell proliferation and motility, cell fate determination, generation of cell polarity  and embryonic inductive interactions.
They do this by regulating and stimulating proliferation along the Notch-Wnt signaling pathway.
Wntprotiens grouped into canonical and non-canonical.
Wnt molecules can bind to the receptors LRP5, Frizzleds, Ror2 and RYK at cell surfaces to activate intracellular signaling pathways.TS19 mouse embryo with areas of Wnt signaling visible in redTargeted knockout of the Wnt4 genes in a female mouse shows that the kidney fails to develop.Wnt signals induce cancerous changes.
Wnt Signaling PathwayNon-canonicalCanonicalCa 2+PCPB-cateninOsteoblast cell proliferation
Cell fate determination
General bone maintenance
Body axis specification
Cancer
intracellular Ca2+ conc.Activation of JNKAffect cytoskeletalorganisation
Ca 2+PCPB-catenin
WNT 4 (Wingless-type MMTV integration site family, member 4):Wnt-4 has the distinction of being the first signaling molecule known to influence sex-determination by participating in gonadal formation . This gene is essential for female development and for suppressing the male reproductive system.
 As a mature molecule, human Wnt-4 is a 46 kDa, 329 aa glycoprotein that contains 24 cysteine residues and two possible N-linked glycosylation sites.
 Wnt-4 expression in mesonephric and gonadalmesenchyme promotes the development of the Müllerianduct (a structure present in the embryo that develops into the uterus, fallopian tubes, cervix, and the upper part of the vagina) and blocks the development of Leydig cells.
 In the same region, Wnt-4 expressing mesenchyme induces a mesenchyme-to-epithelium transition that promotes nephrondevelopment.
In the embryonic adrenal cortex mesoderm, Wnt-4 expression contributes to the proper formation of the zonaglomerulosa, a region that secretes aldosterone.
 Wnt-4 is also found in virgin mammary epithelium. Progesterone increases Wnt-4 expression, leading to an enhanced side-branching (but not elongation) of mammary ducts.
Wnt-4 is detected in fibroblasts in areas of wound healing where fibrin degradation products are abundant.The WNT4 gene is located on the short (p) arm of chromosome 1 between positions 36.23 and 35.1.
WNT4 – A PTH ANABOLIC MEDIATORPTH induces WNT4 mRNA expression in osteoblastic cells 8 hours after PTH injection and maximum expression is observed in the mineralization phase. Serum Ionized Ca2+ level decreasesPTH ReleaseParathyroid GlandEnhance Osteoblast DifferentiationThrough Non-canonical Wnt/Ca2+ and Wnt/PCP pathwayPKCCell Proliferation & CFU-F numbersRegulates WNT4PKAPTH binds to PTHR-1 in Bone, Kidney and other target tissuesExpressed in chondrocytes & mature osteobalstsBy inhibiting BMSSCs apoptosis & stimulating their rate of growth
WNT4-AN ENHANCER OF STEM CELL OSTEOGENIC DIFFERENTIATIONWNT4 , a noncanonical member, was found to potently enhance osteogenic differentiation of MSCs isolated from craniofacial tissues in vitro and bone formation in vivo.Interestingly, Wnt4 did not increase the cytosolic level of B-catenin. But activated a novel noncanonical signaling pathway, P38MAPK, which is known to positively regulate osteogenic differentiation induced by BMPs and other growth factorsWnt 4Stimulates osteocalcin mRNAPrimary osteoblastsPCP & Cgmp/Ca2+ PathwaySome control in osteoblast differentiationProliferating mBMSSCsB-catenin PathwayWnt 4MouseBMSSCsAffects proliferation & differentiationDifferentiating mBMSSCsCa2+ Pathway
In uncommitted MSCs, Wnt4 stimulates B-catenin pathway inducing proliferation.
In committed osteoblasts, it stimulates non-canonical pathways responsible for its effect on differentiation.
Duality of Wnt4’s action depends on Development stage of the cellActive proliferatio of the cell+WNT4mBMSSCsBoth Osteogenic & non-osteogenicconditionsOsteocalcin Bone Marker gene expressionAdipocyte Marker Genes
Specific AimsAim 1 : Assess the Wnt-4 effect on proliferation of human bone marrow stromal stem cells.Aim 2 : Assess the Wnt-4 effect on differentiation of human bone marrow stromal stem cells.Experimental DesignControl and WNT-4 conditioned media collection:Nih3t3 Control Cells grown in DMEM supplemented with 10%FBS & 1%p/sMedium collected @ 80% confluencyAfter syringe filtrationNih3t3 Wnt4 Producing Cells grown in DMEM supplemented with 10%FBS & 1%p/sMedium collected @ 80% confluencyAfter syringe filtrationStored @-80C
Culturing Human BMSSCs with Recombinant WNT4Medium: 10%FBS Alpha-MEM+ 1% p/s+1%Glu+DexIsolate hBMSSCs24ng/ml Wnt4 added to the wnt4 wellsHuman Cancellous Bone SpecimenPlate cells at a conc of 1 million to 3 million cells/well in a 12 well plateLeft untouched for 5 days for the cells to adhereDay 12: Viable Cell Count & Picture TakenDay 5: Medium changed to contain 1 μg/ml Ascorbic AcidMedium Changed every 2-3 days
Control hBMSSCShBMSSCs treated with Wnt4
SIRT1 (Class III HDACs) ProjectSirtuin1, also known as NAD-dependent deacetylasesirtuin-1 stands for sirtuin (silent mating type information regulation 2 homolog) 1.
Sirtuin 1 is a member of the sirtuin family of proteins, homologsof the Sir2 gene in S. cerevisiae.
Sirt 1 is highly expressed in the foetal and adult brain .

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Presentation On Wnt 4 and rhe role of HDAC4 & SIRT1 in bone biology

  • 1. GAYATHRI VIJAYAKUMARDr. Nicola C. PartridgeDept of Basic Science & Craniofacial BiologyNYU-CD
  • 2. WNT PROTEIN FAMILY:Wnt proteins are a family of 19highly conserved cysteine-rich secreted glyco-lipoproteins (39-46 Kda) that regulate development, cell proliferation and motility, cell fate determination, generation of cell polarity  and embryonic inductive interactions.
  • 3. They do this by regulating and stimulating proliferation along the Notch-Wnt signaling pathway.
  • 4. Wntprotiens grouped into canonical and non-canonical.
  • 5. Wnt molecules can bind to the receptors LRP5, Frizzleds, Ror2 and RYK at cell surfaces to activate intracellular signaling pathways.TS19 mouse embryo with areas of Wnt signaling visible in redTargeted knockout of the Wnt4 genes in a female mouse shows that the kidney fails to develop.Wnt signals induce cancerous changes.
  • 6. Wnt Signaling PathwayNon-canonicalCanonicalCa 2+PCPB-cateninOsteoblast cell proliferation
  • 11. intracellular Ca2+ conc.Activation of JNKAffect cytoskeletalorganisation
  • 13. WNT 4 (Wingless-type MMTV integration site family, member 4):Wnt-4 has the distinction of being the first signaling molecule known to influence sex-determination by participating in gonadal formation . This gene is essential for female development and for suppressing the male reproductive system.
  • 14.  As a mature molecule, human Wnt-4 is a 46 kDa, 329 aa glycoprotein that contains 24 cysteine residues and two possible N-linked glycosylation sites.
  • 15.  Wnt-4 expression in mesonephric and gonadalmesenchyme promotes the development of the Müllerianduct (a structure present in the embryo that develops into the uterus, fallopian tubes, cervix, and the upper part of the vagina) and blocks the development of Leydig cells.
  • 16.  In the same region, Wnt-4 expressing mesenchyme induces a mesenchyme-to-epithelium transition that promotes nephrondevelopment.
  • 17. In the embryonic adrenal cortex mesoderm, Wnt-4 expression contributes to the proper formation of the zonaglomerulosa, a region that secretes aldosterone.
  • 18.  Wnt-4 is also found in virgin mammary epithelium. Progesterone increases Wnt-4 expression, leading to an enhanced side-branching (but not elongation) of mammary ducts.
  • 19. Wnt-4 is detected in fibroblasts in areas of wound healing where fibrin degradation products are abundant.The WNT4 gene is located on the short (p) arm of chromosome 1 between positions 36.23 and 35.1.
  • 20. WNT4 – A PTH ANABOLIC MEDIATORPTH induces WNT4 mRNA expression in osteoblastic cells 8 hours after PTH injection and maximum expression is observed in the mineralization phase. Serum Ionized Ca2+ level decreasesPTH ReleaseParathyroid GlandEnhance Osteoblast DifferentiationThrough Non-canonical Wnt/Ca2+ and Wnt/PCP pathwayPKCCell Proliferation & CFU-F numbersRegulates WNT4PKAPTH binds to PTHR-1 in Bone, Kidney and other target tissuesExpressed in chondrocytes & mature osteobalstsBy inhibiting BMSSCs apoptosis & stimulating their rate of growth
  • 21. WNT4-AN ENHANCER OF STEM CELL OSTEOGENIC DIFFERENTIATIONWNT4 , a noncanonical member, was found to potently enhance osteogenic differentiation of MSCs isolated from craniofacial tissues in vitro and bone formation in vivo.Interestingly, Wnt4 did not increase the cytosolic level of B-catenin. But activated a novel noncanonical signaling pathway, P38MAPK, which is known to positively regulate osteogenic differentiation induced by BMPs and other growth factorsWnt 4Stimulates osteocalcin mRNAPrimary osteoblastsPCP & Cgmp/Ca2+ PathwaySome control in osteoblast differentiationProliferating mBMSSCsB-catenin PathwayWnt 4MouseBMSSCsAffects proliferation & differentiationDifferentiating mBMSSCsCa2+ Pathway
  • 22. In uncommitted MSCs, Wnt4 stimulates B-catenin pathway inducing proliferation.
  • 23. In committed osteoblasts, it stimulates non-canonical pathways responsible for its effect on differentiation.
  • 24. Duality of Wnt4’s action depends on Development stage of the cellActive proliferatio of the cell+WNT4mBMSSCsBoth Osteogenic & non-osteogenicconditionsOsteocalcin Bone Marker gene expressionAdipocyte Marker Genes
  • 25. Specific AimsAim 1 : Assess the Wnt-4 effect on proliferation of human bone marrow stromal stem cells.Aim 2 : Assess the Wnt-4 effect on differentiation of human bone marrow stromal stem cells.Experimental DesignControl and WNT-4 conditioned media collection:Nih3t3 Control Cells grown in DMEM supplemented with 10%FBS & 1%p/sMedium collected @ 80% confluencyAfter syringe filtrationNih3t3 Wnt4 Producing Cells grown in DMEM supplemented with 10%FBS & 1%p/sMedium collected @ 80% confluencyAfter syringe filtrationStored @-80C
  • 26. Culturing Human BMSSCs with Recombinant WNT4Medium: 10%FBS Alpha-MEM+ 1% p/s+1%Glu+DexIsolate hBMSSCs24ng/ml Wnt4 added to the wnt4 wellsHuman Cancellous Bone SpecimenPlate cells at a conc of 1 million to 3 million cells/well in a 12 well plateLeft untouched for 5 days for the cells to adhereDay 12: Viable Cell Count & Picture TakenDay 5: Medium changed to contain 1 μg/ml Ascorbic AcidMedium Changed every 2-3 days
  • 28. SIRT1 (Class III HDACs) ProjectSirtuin1, also known as NAD-dependent deacetylasesirtuin-1 stands for sirtuin (silent mating type information regulation 2 homolog) 1.
  • 29. Sirtuin 1 is a member of the sirtuin family of proteins, homologsof the Sir2 gene in S. cerevisiae.
  • 30. Sirt 1 is highly expressed in the foetal and adult brain .
  • 31. Sirt1 not only deacetylateshistones H1, H3 and H4, but also deacetylates many non-histone proteins including p53, FOXO, Ku70, p300, Rb, E2F1, NF-kB, p73 and PGC-1α .
  • 32. In virtue of these important targets, SIRT1 is linked to regulatory control of diverse normal and abnormal cellular processes ranging from stress responses, aging, and metabolism to cancer.
  • 33. It  is down-regulated in human senescent cells, suggesting that SIRT1 may be required to extend replicative life span 
  • 34.  It   deacetylated   p53,thus antagonizing  p53 mediated apoptosis and increasing cell survival. Cancer cell lines revealed  higher endogenous level of SIRT1 expression compared to normal cells .HDAC4:Histonedeacetylases (HDACs) are transcriptional coregulators with the capability of modifying chromatin structure and other transcription factors.
  • 35. HDACs play a role in deacetylation of lysine residues in the tails of core histones.
  • 36. They are classified into 4 groups.
  • 37. HDAC4 binding sites are three serine residues, which are calcium-calmodulin –dependent kinase (CaMK) phosphorylation sites.C-Fos:c-Fosis a cellular proto-oncogene belonging to the immediate early gene family of transcription factors. c-Fos has a leucine-zipperDNA binding domain, and a transactivation domain at the C-terminus. C-Jun:c-Jun is the name of a gene and protein that, in combination with c-Fos, forms the AP-1 early response transcription factor.
  • 38.  It is activated through double phosphorylation by the JNK pathway but has also a phosphorylation-independent function.
  • 39. It is a proto-oncogene as well and belongs to the IEG family.PTH Induced Bone Resorption & Bone RemodelingPTH regulates calcium metabolism by acting on osteoblasts to produce Osteoclast Activating Factors such as RANKL, MMP-13, or Collagenase-3.PTH induces MMP-13 gene transcription through a PKA dependent pathway.
  • 40. HDAC-4Under Basal ConditionRunx2RDAP-1MMP-13 PromoterPTH EffectHDAC-4Runx2PKA dependent phosphorylationPTHRDAP-1MMP-13 PromoterCytoplasmRunx2JUN & FOSpcafp300HATpcafp300RDAP-1HATNucleus
  • 42. AIM:Investigating the role of Sirt-1 in regulating the expression of MMP-13.Establishing the characteristics of SIRT1 association with AP-1 proteins in PTH-treated Osteoblasts.
  • 43. Whether there is association of Sirt-1 with Fos or Jun in Osteoblastic cells.
  • 44. Whether there is an interaction between HDAC4 and Sirt1 as well as between HDAC4 , Fos and Jun.EXPERIMENT:Preparation of cell lysatePTH treatment for 4 and 8 hoursUMR cells cultured till 80-90% confluentImmunopercipitated for Sirt1, Fos, Jun or HDAC4Immunoblot for Sirt-1, HDAC4, Fosor Jun
  • 45. Immunoblot Sirt-1IgGjunFosIPC4 P4 C8 P8 C4 P4 C8 P8 C4 P4 C8 P8Immunoblot Sirt-1IgGSirt-1HDAC4IP C4 P4 C8 P8 C4 P4C8 P8 C4 P4 C8 P8
  • 46. Immunoblot HDAC4IgGjunFosIPC4 P4 C8 P8 C4 P4 C8 P8 C4 P4 C8 P8Immunoblot HDAC4IgGSirt-1HDAC4IPC4 P4 C8 P8 C4 P4 C8 P8 C4 P4 C8 P8
  • 47. THE ORTHOFIX PROJECTPULSED ELECTROMAGNETIC FIELD THERAPY:Pulsed Electromagnetic Field Therapy (PEMF), also called Pulsed Magnetic Therapy, is a reparative technique most commonly used in the field of orthopedics for the treatment of non-union fractures, failed fusions, and congenital pseudarthrosis. HISTORY:In1970s,the FDA approved the use of a specific biphasic low frequency signal for the treatment of non-union/delayed fractures –introduced by the Andrew Basset team.
  • 48. A decade later, FDA allowed the use of pulsed radiofrequency electromagnetic field (PRF) for treatment of pain and edema in superficial soft tissues.Three biological windows have been identified by analyzing the response of cells to a range of amplitudes and frequencies. These are:PEMF50–100 lT (5–10 Gauss)15–20 mT (150–200 Gauss) and45–50 mT (450–500 Gauss) (Markov 2005).
  • 49. NASA Signal Driver (Hydra) Project ProtocolRat Primary osteoblasts isolated from postnatal day 1 rat calvariaePlated with MEM+10% FBS. Treated with the PEMF signal 4h a day. Control not treated.Day 6 (proliferation stage) : Cell number countedDay 8: Induction of differentiationRT-PCRDay 12 & Day 18: Total RNA isolated from cells & von Kossa StainingMedium switch with BGJ + 10%FBS + Ascorbic Acid + ß-glycerophosphate
  • 50. Genes Analyzed in the NASA-ORTHOFIX Project 1.Osteocalcinalso known as bone gamma-carboxyglutamic acid-containing protein (BGLAP), is a noncollagenous protein secreted solely by osteoblasts.It is used as a biomarker for bone formation2.Bone Alkaline Phosphatase (BAP)It is secreted by osteoblasts. Alkaline Phosphatase (ALP) is a ubiquitous enzyme associated with cell membranes.. It is produced by osteoblasts to provide a high PO4 concentration at the osteoblast cell surface during bone mineralization and is a marker of bone formation.3.Type I Collagenformed by osteoblasts; reflects rate of collagen and bone formation; most sensitive marker of bone formation and particularly useful for monitoring bone formation therapies and antiresorptive therapies.4.Glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) is an enzyme that catalyzes the sixth step of glycolysis besides transcription activation, initiation of apoptosis, and ER to Golgi vesicle shuttling. Because the GAPDH gene is often stably and constitutively expressed at high levels in most tissues and cells, it is considered a housekeeping gene.
  • 51. Osteoblast Mineralization – von Kossa StainingCells fixed with 95% Ethanol (15 min at 37°C)Rinsed with 80% EthanolRinsed with 50% EthanolDay 12 &18 Incubated with 5% silver nitrate solution (1 hr at 37°C) Rinsed with 20% EthanolWashed with waterWashed with waterMeasured by Image QuantUV Light (10 mins)Dried and Photographed
  • 52. Overall Fold Stimulation in Cell Count during Proliferation Stage
  • 53. Overall Fold Stimulation for Differentiation Stage
  • 54. Overall Fold Stimulation for Mineralization Stage
  • 55. Von Kossa Staining – Differentiation StageControlPEMF Treated
  • 56. Image Quant Data for von Kossa Staining- Differentiation Stage
  • 57. Von Kossa Staining – Mineralization StageControlPEMF Treated
  • 58. Image Quant Data for von Kossa Staining- Mineralization Stage