title: Chondrogenic activity of the heparan sulfate proteoglycan perlecan maps to the N-terminal domain I.
abstract: C3H10T1/2 cells differentiate along a chondrogenic pathway when plated onto the extracellular matrix (ECM) protein perlecan (Pln). To identify the region(s) within the large Pln molecule that provides a differentiation signal, recombinant Pln-sequence-based polypeptides representing distinct structural domains were assayed for their ability to promote chondrogenesis in C3H10T1/2 cells. Five distinct domains, along with structural variations, were tested. The N-terminal domain I was tested in two forms (IA and IB) that contain only heparan sulfate (HS) chains or both HS and chondroitin sulfate (CS) chains, respectively. A mutant form of domain I lacking attachment sites for both HS and CS (Pln I(mut)) was tested also. Other constructs consecutively designated Pln domains II, III(A-C), IV(A,B), and V(A,B) were used to complete the structure-function analysis. Cells plated onto Pln IA or Pln IB but no other domain rapidly assembled into cellular aggregates of 40-120 microm on average. Aggregate formation was dependent on the presence of glycosaminoglycan (GAG) chains, because Pln I-based polypeptides lacking GAG chains either by enzymatic removal or mutation of HS/CS attachment sites were inactive. Aggregates formed on GAG-bearing Pln IA stained with Alcian Blue and were recognized by antibodies to collagen type II and aggrecan but were not recognized by an antibody to collagen type X, a marker of chondrocyte hypertrophy. Collectively, these studies indicate that the GAG-bearing domain I of Pln provides a sufficient signal to trigger C3H10T1/2 cells to enter a chondrogenic differentiation pathway. Thus, this matrix proteoglycan (PG) found at sites of cartilage formation in vivo is likely to enhance early stage differentiation induced by soluble chondrogenic factors.
authors.affiliation: Graduate School of Biomedical Sciences, University of Texas, Houston, USA.
authors.forename: Margaret M
authors.initials: MM
authors.lastname: French
authors.forename: Ronald R
authors.initials: RR
authors.lastname: Gomes
authors.forename: Rupert
authors.initials: R
authors.lastname: Timpl
authors.forename: Magnus
authors.initials: M
authors.lastname: Höök
authors.forename: Kirk
authors.initials: K
authors.lastname: Czymmek
authors.forename: Mary C
authors.initials: MC
authors.lastname: Farach-Carson
authors.forename: Daniel D
authors.initials: DD
authors.lastname: Carson
mesh_terms: D000818:Animals; D001665:Binding Sites / Q000235:genetics; D002449:Cell Aggregation; D002454:Cell Differentiation; D002460:Cell Line; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism; D020219:Chondrogenesis / Q000235:genetics / Q000502:physiology*; D002809:Chondroitin Sulfates / Q000737:chemistry; D006025:Glycosaminoglycans / Q000737:chemistry; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D006497:Heparan Sulfate / Q000737:chemistry; D051379:Mice; D009154:Mutation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics
chemical_list: D002809:Chondroitin Sulfates; D006025:Glycosaminoglycans; D019812:Heparan Sulfate Proteoglycans; D006497:Heparan Sulfate; D011994:Recombinant Proteins; C071927:perlecan