MKP‐1 LPS-Driven Osteoclastogenesis More Prevalent in Dusp‐1‐/‐ Females Than Males

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Browne, Courtney G
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Periodontal disease is the result of chronic inflammatory bone loss due to a sustained immune system response to pathogenic invasion. Osteoclasts (OC) are multinucleated, bone resorbing cells that play a vital role in bone turnover. Receptor activator of nuclear factor kappa B ligand (RANKL) is the physiological regulator of osteoclastogenesis (OCgen) in naïve progenitor populations. Gram-negative bacteria involved in pathogenesis of periodontal disease contain the endotoxin, lipopolysaccharide (LPS), which increases RANKL expression by activating innate immune inflammatory cascades including mitogen activated protein kinase (MAPK). MAPK activity is negatively regulated by MAPK phosphatase (MKP)-1 which is encoded by the gene Dusp-1. The current study aims to investigate the role of Dusp-1 when driven by LPS and primed with RANKL OC progenitors (OCP). Based on the role of MKP-1 as a negative regulator of activated MAPK, we hypothesize that Dusp-1 deficient OCP will form more OCs in response to LPS stimuli. OCPs were isolated from male and female hematopoietic stem cells via magnet-activated cell sorting (MACS) and plated (5x104/well) based on expression of CD11b (CD11bhigh, low and neg), from Dusp-1+/+ and Dusp-1-/- mice. Cells were primed with macrophage colony stimulating factor (M-CSF) and RANKL for 48 hours and were then stimulated with 10ng/ml of A. actinomycetemcomitans LPS for up to 4 days. A tartrate resistant acid phosphatase (TRAP) assay was used to visualize formation of OCs at day 0 (M-CSF +RANKL only) and days 2 and 4 post-LPS. Images generated from TRAP were used to determine the number of OCs, number of nuclei per OC, and area per OC. Day 0 cells lacked any exposure to LPS, resulting in minimal variation between Dusp-1+/+ and Dusp-1-/- for all CD11b populations. By Day 2 there was a significant trend of increased female Dusp-1-/- OCgen, with CD11bhigh having the greatest difference (p<0.05). Male data followed an opposite trend, with Dusp-1-/- having less OCgen for all CD11b sorted populations. By Day 4, female CD11bhigh (p<0.0001) and CD11blow (p<0.05) had significantly more OCs for Dusp-1-/-. Day 4 of the males showed a large decrease in Dusp-1+/+ cells, specifically in CD11blow. The data show that LPS induced more osteoclast formation in female Dusp-1-/- mice than Dusp-1+/+ mice. The male Dusp-1+/+ populations show a preliminary increase in OCgen. However, by day 4 there are substantially more Dusp-1-/- cells that are larger than their Dusp1+/+ counterparts. Therefore, these studies indicate that MKP-1 is key negative regulator of MAPK and the subsequent increased OC differentiation promotes inflammatory bone resorption.
MKP-1, LPS, RANKL, osteoclastogenesis, periodontal disease, Dusp-1