Cyclosporin A affects extracellular matrix synthesis and degradation by mouse MC3T3-E1 osteoblasts in vitro

Alessia Fornoni, Flavia Cornacchia, Guy Howard, Bernard A. Roos, Gary E. Striker, Liliane J. Striker

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Background. Immunosuppressant therapy is thought to be a major contributor to post-transplant bone disease. Histological data and serum parameters suggest that Cyclosporin A (CsA) treatment causes osteopenia as a result of an altered bone turnover, but the pathogenic mechanisms of this process remain unclear. We investigate if CsA affects cell turnover and extracellular matrix (ECM) synthesis and degradation in MC3T3-E1 osteoblasts, as a surrogate model for in vivo events. Methods. Cell were exposed to increasing doses of CsA (0, 0.5, 1 and 5 μg/ml). Proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation, viability by Trypan Blue exclusion and apoptosis by ELISA. Type I collagen was measured by ELISA and reverse transcription-polymerase chain reaction (RT-PCR), matrix metalloproteinases (MMP) by zymography and RT-PCR, and tissue inhibitors of MMP (TIMP) by reverse zymography. Results. CsA exposure for 48 h decreased osteoblast-number in a dose-dependent manner in the absence of apoptosis or cytotoxicity. CsA at a dose of 5 μg/ml for 72 h caused decreased collagen type I mRNA expression and protein accumulation. While MMP-2 remained unaffected, MMP-9 activity increased. TIMP-1 activity was unaffected, while a dose-dependent increase of TIMP-2 was observed. Conclusions. These data suggest that CsA alters ECM synthesis and degradation in MC3T3-E1 osteoblasts by decreasing type I collagen production and increasing MMP-9 activity. The combination of increased MMP-9 with unchanged TIMP-1 activity could reduce the osteoid matrix available for mineralization. In addition, decreased proliferation could further reduce the number of cells synthesizing new osteoid matrix and thus contribute to the process of bone loss.

Original languageEnglish
Pages (from-to)500-505
Number of pages6
JournalNephrology Dialysis Transplantation
Volume16
Issue number3
StatePublished - Apr 2 2001

Fingerprint

Osteoblasts
Cyclosporine
Extracellular Matrix
Matrix Metalloproteinase Inhibitors
Matrix Metalloproteinase 9
Collagen Type I
Reverse Transcription
Enzyme-Linked Immunosorbent Assay
Apoptosis
Tissue Inhibitor of Metalloproteinases
Polymerase Chain Reaction
Trypan Blue
Bone Remodeling
Metabolic Bone Diseases
Bone Diseases
Matrix Metalloproteinase 2
Bromodeoxyuridine
Immunosuppressive Agents
Matrix Metalloproteinases
In Vitro Techniques

Keywords

  • Cyclosporine
  • Extracellular matrix
  • Osteoblasts

ASJC Scopus subject areas

  • Nephrology
  • Transplantation

Cite this

Cyclosporin A affects extracellular matrix synthesis and degradation by mouse MC3T3-E1 osteoblasts in vitro. / Fornoni, Alessia; Cornacchia, Flavia; Howard, Guy; Roos, Bernard A.; Striker, Gary E.; Striker, Liliane J.

In: Nephrology Dialysis Transplantation, Vol. 16, No. 3, 02.04.2001, p. 500-505.

Research output: Contribution to journalArticle

Fornoni, Alessia ; Cornacchia, Flavia ; Howard, Guy ; Roos, Bernard A. ; Striker, Gary E. ; Striker, Liliane J. / Cyclosporin A affects extracellular matrix synthesis and degradation by mouse MC3T3-E1 osteoblasts in vitro. In: Nephrology Dialysis Transplantation. 2001 ; Vol. 16, No. 3. pp. 500-505.
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AB - Background. Immunosuppressant therapy is thought to be a major contributor to post-transplant bone disease. Histological data and serum parameters suggest that Cyclosporin A (CsA) treatment causes osteopenia as a result of an altered bone turnover, but the pathogenic mechanisms of this process remain unclear. We investigate if CsA affects cell turnover and extracellular matrix (ECM) synthesis and degradation in MC3T3-E1 osteoblasts, as a surrogate model for in vivo events. Methods. Cell were exposed to increasing doses of CsA (0, 0.5, 1 and 5 μg/ml). Proliferation was evaluated by bromodeoxyuridine (BrdU) incorporation, viability by Trypan Blue exclusion and apoptosis by ELISA. Type I collagen was measured by ELISA and reverse transcription-polymerase chain reaction (RT-PCR), matrix metalloproteinases (MMP) by zymography and RT-PCR, and tissue inhibitors of MMP (TIMP) by reverse zymography. Results. CsA exposure for 48 h decreased osteoblast-number in a dose-dependent manner in the absence of apoptosis or cytotoxicity. CsA at a dose of 5 μg/ml for 72 h caused decreased collagen type I mRNA expression and protein accumulation. While MMP-2 remained unaffected, MMP-9 activity increased. TIMP-1 activity was unaffected, while a dose-dependent increase of TIMP-2 was observed. Conclusions. These data suggest that CsA alters ECM synthesis and degradation in MC3T3-E1 osteoblasts by decreasing type I collagen production and increasing MMP-9 activity. The combination of increased MMP-9 with unchanged TIMP-1 activity could reduce the osteoid matrix available for mineralization. In addition, decreased proliferation could further reduce the number of cells synthesizing new osteoid matrix and thus contribute to the process of bone loss.

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