Basic calcium phosphate crystals up-regulate metalloproteinases but down-regulate tissue inhibitor of metalloproteinase-1 and -2 in human fibroblasts.
OBJECTIVE
To examine the effect of basic calcium phosphate (BCP) crystals on expression of tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in human fibroblasts.


METHODS
Using a semi-quantitative reverse transcription-polymerase chain reaction method and phosphocitrate (PC), a specific inhibitor of the biological effects of BCP crystals, we examined the effects of BCP on the steady state transcript levels of metalloproteinase (MMP)-1, -3, -9 and -13 and TIMP-1 and -2 in human fibroblasts. DNA primers against elongation factor were used as internal controls. RNAs isolated from human fibroblasts treated with BCP crystals (50 microg/ml) in the presence or absence of PC (10(-3) M) were used as templates, and RNA from untreated control cultures and cultures treated with Interleukin-1-beta (IL-1beta) were used as negative and positive controls, respectively.


RESULTS
We observed increases in MMP-1, -3, -9 and -13 transcripts by BCP crystals. BCP crystal down-regulated TIMP-1 and -2 over untreated controls. Western blot analysis confirmed that BCP crystals down-regulate the synthesis of TIMP-1 and -2. While IL-1beta up-regulated MMP-1, -3, -9 and -13, it had no significant effect on expression of either TIMP. In all cases, PC specifically reversed the differential regulation of MMPs and TIMPs by BCP crystals but had no effect on IL-1beta induction of MMP expression.


CONCLUSIONS
The ability of BCP to induce the synthesis of degradative MMPs while down-regulating the synthesis of the naturally occurring counterpart TIMPs may explain the changes consistent with a role of BCP crystal in the pathogenesis of degenerative changes in osteoarthritis. The ability of PC to reverse both degradative effects of BCP crystal suggests that PC can be a potential therapeutic agent for BCP crystal deposition diseases.
Bai
G
G
Research Service and Geriatric Research, Education, and Clinical Center, VA Medical Center, Miami, Florida 33125, USA.
Howell
D S
DS
Howard
G A
GA
Roos
B A
BA
Cheung
H S
HS
D015153:Blotting, Western / Q000379:methods; D002130:Calcium Phosphates / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002478:Cells, Cultured; D002951:Citrates / Q000494:pharmacology; D017364:Collagenases / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D020781:Matrix Metalloproteinase 1 / Q000235:genetics / Q000378:metabolism; D053509:Matrix Metalloproteinase 13; D019278:Matrix Metalloproteinase 3 / Q000235:genetics / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D020782:Matrix Metalloproteinases / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000235:genetics / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases / Q000235:genetics / Q000378:metabolism*
D002130:Calcium Phosphates; D002951:Citrates; D007375:Interleukin-1; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D019714:Tissue Inhibitor of Metalloproteinases; C485828:alpha-tricalcium phosphate; C485830:tetracalcium phosphate; D019716:Tissue Inhibitor of Metalloproteinase-2; C014148:phosphocitrate; C485838:calcium phosphate, monobasic, anhydrous; C020243:calcium phosphate; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; D020782:Matrix Metalloproteinases; D019278:Matrix Metalloproteinase 3; D020780:Matrix Metalloproteinase 9; D020781:Matrix Metalloproteinase 1; C485829:calcium phosphate, dibasic, anhydrous