Effects of TGF-β1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures

G. D. DuRaine, S. M T Chan, A Hari Reddi

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Objective: Superficial Zone Protein (SZP) is expressed by the superficial zone chondrocytes and is involved in boundary lubrication of the articular cartilage surface. SZP protein expression is dependent on anatomical location and is regulated by the transforming growth factor-β (TGF-β) pathway. The hypothesis of this study was that between load-bearing, and non-load-bearing locations, of the femoral medial condyle alternative splice isoforms of SZP are different, and regulated by TGF-β1. Methods: Using reverse transcription-polymerase chain reaction (RT-PCR) we identified differentially expressed SZP alternative splicing. Using recombinant proteins of the N-terminal region produced from these isoforms, we identified differences in binding to heparin and the extracellular matrix. Results: We identified a novel splice form of SZP (isoform E), lacking exons 2-5. Differences in alternative splicing were observed between anterior load-bearing locations of the femoral medial condyle (M1) compared to the posterior non-load-bearing location (M4). TGF-β1 increased splicing out of exons 4 and 5 encoding a heparin binding domain. The minimal induction time for changes in splicing by TGF-β1 at the M1 location was 1. h, although this did change total SZP mRNA levels. Inhibition of Smad3 phosphorylation inhibited TGF-β1 induced splicing, and SZP protein expression. Recombinant proteins corresponding to isoforms upregulated by TGF-β1 had reduced binding. The SZP dimerization domain is located within exon 3. Conclusions: In conclusion, alternative splicing of SZP is regulated by TGF-β1 signaling and may regulate SZP interaction with heparin/heparan sulfate or other components in the extracellular matrix of articular cartilage by splicing out of the heparin binding domain.

Original languageEnglish (US)
Pages (from-to)103-110
Number of pages8
JournalOsteoarthritis and Cartilage
Volume19
Issue number1
DOIs
StatePublished - Jan 2011

Fingerprint

Alternative Splicing
Cartilage
Articular Cartilage
Transforming Growth Factors
Proteins
Bearings (structural)
Heparin
Protein Isoforms
Exons
Weight-Bearing
Thigh
Recombinant Proteins
Recombinant proteins
Extracellular Matrix
Protein Multimerization
Protein Splicing
Lubrication
Intercellular Signaling Peptides and Proteins
Bone and Bones
Heparitin Sulfate

Keywords

  • Alternative splicing
  • Articular cartilage
  • Superficial Zone Protein (SZP)
  • TGF-β

ASJC Scopus subject areas

  • Biomedical Engineering
  • Orthopedics and Sports Medicine
  • Rheumatology

Cite this

Effects of TGF-β1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures. / DuRaine, G. D.; Chan, S. M T; Reddi, A Hari.

In: Osteoarthritis and Cartilage, Vol. 19, No. 1, 01.2011, p. 103-110.

Research output: Contribution to journalArticle

@article{45eae8ef64cc4bd290e0de51daa504bf,
title = "Effects of TGF-β1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures",
abstract = "Objective: Superficial Zone Protein (SZP) is expressed by the superficial zone chondrocytes and is involved in boundary lubrication of the articular cartilage surface. SZP protein expression is dependent on anatomical location and is regulated by the transforming growth factor-β (TGF-β) pathway. The hypothesis of this study was that between load-bearing, and non-load-bearing locations, of the femoral medial condyle alternative splice isoforms of SZP are different, and regulated by TGF-β1. Methods: Using reverse transcription-polymerase chain reaction (RT-PCR) we identified differentially expressed SZP alternative splicing. Using recombinant proteins of the N-terminal region produced from these isoforms, we identified differences in binding to heparin and the extracellular matrix. Results: We identified a novel splice form of SZP (isoform E), lacking exons 2-5. Differences in alternative splicing were observed between anterior load-bearing locations of the femoral medial condyle (M1) compared to the posterior non-load-bearing location (M4). TGF-β1 increased splicing out of exons 4 and 5 encoding a heparin binding domain. The minimal induction time for changes in splicing by TGF-β1 at the M1 location was 1. h, although this did change total SZP mRNA levels. Inhibition of Smad3 phosphorylation inhibited TGF-β1 induced splicing, and SZP protein expression. Recombinant proteins corresponding to isoforms upregulated by TGF-β1 had reduced binding. The SZP dimerization domain is located within exon 3. Conclusions: In conclusion, alternative splicing of SZP is regulated by TGF-β1 signaling and may regulate SZP interaction with heparin/heparan sulfate or other components in the extracellular matrix of articular cartilage by splicing out of the heparin binding domain.",
keywords = "Alternative splicing, Articular cartilage, Superficial Zone Protein (SZP), TGF-β",
author = "DuRaine, {G. D.} and Chan, {S. M T} and Reddi, {A Hari}",
year = "2011",
month = "1",
doi = "10.1016/j.joca.2010.10.008",
language = "English (US)",
volume = "19",
pages = "103--110",
journal = "Osteoarthritis and Cartilage",
issn = "1063-4584",
publisher = "W.B. Saunders Ltd",
number = "1",

}

TY - JOUR

T1 - Effects of TGF-β1 on alternative splicing of Superficial Zone Protein in articular cartilage cultures

AU - DuRaine, G. D.

AU - Chan, S. M T

AU - Reddi, A Hari

PY - 2011/1

Y1 - 2011/1

N2 - Objective: Superficial Zone Protein (SZP) is expressed by the superficial zone chondrocytes and is involved in boundary lubrication of the articular cartilage surface. SZP protein expression is dependent on anatomical location and is regulated by the transforming growth factor-β (TGF-β) pathway. The hypothesis of this study was that between load-bearing, and non-load-bearing locations, of the femoral medial condyle alternative splice isoforms of SZP are different, and regulated by TGF-β1. Methods: Using reverse transcription-polymerase chain reaction (RT-PCR) we identified differentially expressed SZP alternative splicing. Using recombinant proteins of the N-terminal region produced from these isoforms, we identified differences in binding to heparin and the extracellular matrix. Results: We identified a novel splice form of SZP (isoform E), lacking exons 2-5. Differences in alternative splicing were observed between anterior load-bearing locations of the femoral medial condyle (M1) compared to the posterior non-load-bearing location (M4). TGF-β1 increased splicing out of exons 4 and 5 encoding a heparin binding domain. The minimal induction time for changes in splicing by TGF-β1 at the M1 location was 1. h, although this did change total SZP mRNA levels. Inhibition of Smad3 phosphorylation inhibited TGF-β1 induced splicing, and SZP protein expression. Recombinant proteins corresponding to isoforms upregulated by TGF-β1 had reduced binding. The SZP dimerization domain is located within exon 3. Conclusions: In conclusion, alternative splicing of SZP is regulated by TGF-β1 signaling and may regulate SZP interaction with heparin/heparan sulfate or other components in the extracellular matrix of articular cartilage by splicing out of the heparin binding domain.

AB - Objective: Superficial Zone Protein (SZP) is expressed by the superficial zone chondrocytes and is involved in boundary lubrication of the articular cartilage surface. SZP protein expression is dependent on anatomical location and is regulated by the transforming growth factor-β (TGF-β) pathway. The hypothesis of this study was that between load-bearing, and non-load-bearing locations, of the femoral medial condyle alternative splice isoforms of SZP are different, and regulated by TGF-β1. Methods: Using reverse transcription-polymerase chain reaction (RT-PCR) we identified differentially expressed SZP alternative splicing. Using recombinant proteins of the N-terminal region produced from these isoforms, we identified differences in binding to heparin and the extracellular matrix. Results: We identified a novel splice form of SZP (isoform E), lacking exons 2-5. Differences in alternative splicing were observed between anterior load-bearing locations of the femoral medial condyle (M1) compared to the posterior non-load-bearing location (M4). TGF-β1 increased splicing out of exons 4 and 5 encoding a heparin binding domain. The minimal induction time for changes in splicing by TGF-β1 at the M1 location was 1. h, although this did change total SZP mRNA levels. Inhibition of Smad3 phosphorylation inhibited TGF-β1 induced splicing, and SZP protein expression. Recombinant proteins corresponding to isoforms upregulated by TGF-β1 had reduced binding. The SZP dimerization domain is located within exon 3. Conclusions: In conclusion, alternative splicing of SZP is regulated by TGF-β1 signaling and may regulate SZP interaction with heparin/heparan sulfate or other components in the extracellular matrix of articular cartilage by splicing out of the heparin binding domain.

KW - Alternative splicing

KW - Articular cartilage

KW - Superficial Zone Protein (SZP)

KW - TGF-β

UR - http://www.scopus.com/inward/record.url?scp=78650308613&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78650308613&partnerID=8YFLogxK

U2 - 10.1016/j.joca.2010.10.008

DO - 10.1016/j.joca.2010.10.008

M3 - Article

C2 - 20955806

AN - SCOPUS:78650308613

VL - 19

SP - 103

EP - 110

JO - Osteoarthritis and Cartilage

JF - Osteoarthritis and Cartilage

SN - 1063-4584

IS - 1

ER -