Background/Purpose: Hemochromatosis (HH) is one of the strongest known risk factors for calcium pyrophosphate (CPP) crystal deposition. The pathogenic mechanisms causing CPP crystal formation in HH patients remain unknown. CPP crystal formation is associated with increased levels of inorganic pyrophosphate (PPi). This process is controlled by transporters of extracellular ATP such as ANK, and a series of ectoenzymes that hydrolyze ATP or degrade PPi. Recently, alterations in bone such as increased osteoclastogenesis, have also been implicated in CPPD. Most end organ damage in HH is attributed to iron deposition; and in vitro studies mimic HH by exposing cells or tissues to high levels of iron. Prior work showed no clear effects of iron on factors affecting CPP crystal formation in articular cartilage. We sought to investigate the hypothesis that iron contributes to CPP crystal formation by stimulating osteoclastogenesis. We also explored other effects of iron on PPi regulatory factors in osteoclasts and osteoblasts.

Methods: RAW264.7 cells (pre-osteoclasts) were incubated with 10-20 ng/ml RANKL and 10-100 µM ferric ammonium citrate (FAC) or no additives for 4-6 days. Osteoclast formation was assessed using a quantitative TRAP assay. Many of the effects of iron are attributed to increased reactive oxygen species (ROS). We measured ROS in RAW cells using DCFDA dye (Sigma). Because high levels of RANKL on osteoblasts stimulate osteoclast formation, we measured RANKL on osteoblasts in the presence of iron. Human osteoblasts (fOBs, Cell Applications) were exposed to 10-100 µM FAC or no additives for 9 days. RANKL levels were measured using RT-PCR. Other factors known to contribute to bone PPi homeostasis were measured in fOBs, including alkaline phosphatase (AP) activity, ANK levels, and levels of PPi. ANK was measured with RT-PCR and AP activity was measured with an assay from Abcam. PPi was measured in the conditioned media of fOBs and RAW cells using a highly sensitive radiometric assay.

Results: Ten-40 µM FAC significantly increased TRAP activity on RAW cells incubated with submaximal concentrations of RANKL at 6 days (p< 0.0001, N=15). Forty µM FAC increased TRAP activity by 2-fold (Figure 1). FAC also increased ROS (p< 0.0001, N=5) (Figure 2). Similar concentrations of FAC had no effects on fOB RANKL levels at 9 days in the absence or presence of differentiation factors (Data not shown). There was a small but significant decrease in alkaline phosphatase in fOBs at 100 µM FAC. Levels were 4.03± 1.3 nmol/well in fOBs with no iron exposure and 2.35± 0.6 U with FAC (p=0.02, n=5). FAC had no effects on ANK mRNA, and PPi levels were unmeasurable in both RAWs and fOBs. FAC toxicity was monitored using an LDH assay and was not significant at the concentrations tested.

Conclusion: We confirm here that iron increases TRAP positive osteoclasts in vitro. Increased osteoclast number is associated with premature severe CPPD caused by a loss of function mutation in osteoprotegerin. Further work will be necessary to determine the mechanisms through which osteoclast excess causes CPPD, but these findings add additional support to the hypothesis that some forms of CPDD are related to primary abnormalities in bone.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/hemochromatosis-is-associated-with-cppd-through-irons-effect-on-bone/