Abstract
Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.
Highlights
Bone loss is a natural phenomenon of the aging process
In this study we evaluated the functions of Phlpp1 within Cathepsin K (Ctsk)-expressing cells in a model of enhanced bone resorption
Phlpp1 cKOCtsk mice lose an equivalent amount of bone to their control littermates following ovariectomy
Summary
Bone loss is a natural phenomenon of the aging process. As both men and women age, there is a relative imbalance in bone resorption as compared to formation which leads to negative. Age-related decline disrupts the reversal phase, leading to uncoupling of bone resorption and bone formation phases This creates an imbalance between the amount of bone resorbed compared to that formed with each remodeling cycle. The compounded effects of this whole process are exacerbated by an increase in the frequency with which new remodeling cycles are activated after the menopause This leads to postmenopausal acceleration of bone loss [4]. Conditional deletion of Phlpp in Cathepsin K (Ctsk)-Cre-expressing cells resulted in enhancement of bone mass characterized by diminished osteoclast activity and enhanced coupling to bone formation [7]. Due to the limitations of the Ctsk-Cre driver, including expression within mesenchymal lineage cells [8,9,10,11,12], we further explored the functions of Phlpp in a model of enhanced bone resorption. This study demonstrates that Phlpp limits expression of Ifgbp, known to be required for optimal bone mass attainment in females [13], and that this repression is lost following ovariectomy
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