Cyclosporin A affects extracellular matrix synthesis and degradation by mouse MC3T3-E1 osteoblasts in vitro.
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
Cells were exposed to increasing doses of CsA (0, 0.5, 1 and 5 microg/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 microg/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.
Renal Cell Biology Laboratory and Vascular Biology Institute, University of Miami School of Medicine, Miami, Florida 33101, USA.
A
A
Fornoni
F
F
Cornacchia
G A
GA
Howard
B A
BA
Roos
G E
GE
Striker
L J
LJ
Striker
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003094:Collagen / Q000037:antagonists & inhibitors / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005109:Extracellular Matrix / Q000378:metabolism*; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism
D007166:Immunosuppressive Agents; D012333:RNA, Messenger; D019716:Tissue Inhibitor of Metalloproteinase-2; D016572:Cyclosporine; D003094:Collagen; D000469:Alkaline Phosphatase; D020780:Matrix Metalloproteinase 9