Full Length ArticleSelective inhibition of progesterone receptor in osteochondral progenitor cells, but not in mature chondrocytes, modulated subchondral bone structures
Introduction
The presence or relative proportion of progesterone nuclear receptors (PR) in different tissues may contribute to sexual dimorphism in these tissues. The function of the PR in chondrocytes is mostly unknown. Over the past ten years, our research group and others have studied PR action on the skeleton. PR is expressed by cultured osteoblasts, osteoclasts [[1], [2], [3]], and chondrocytes [4] and is present in vivo in mouse bone [3,5]. Both genetic fate mapping and immunohistochemistry have identified PR (esp. PR-B) expression in subchondral cartilage and the chondrocytes in the growth plate [5]. Additionally, conditional PR deletion in osteochondrogenic progenitor cells significantly suppressed immunomodulation pathways that might affect disease pathways involved in the development of osteoarthritis (OA) [6].
OA is characterized by degenerative changes in the whole joint leading to pain and ultimately physical disability. The risk of developing OA increases in post-menopausal women [7,8], suggesting a link between OA and hormonal status, which has traditionally been attributed to changes in estrogen levels. Women with low circulating estradiol levels were nearly twice as likely to develop OA or experience joint pain as those with higher hormone levels, after adjusting for age, injury history, and body-mass index [9]. Long-term estrogen replacement therapy might provide a moderate level of protection against knee OA [8,[10], [11], [12]] which might be due to attenuation of pro-inflammatory cytokines resulting from estrogen deficiency [8,13]. Gene polymorphisms in the estrogen receptors (ERs) α & β have been found to be associated with knee and hand OA [[14], [15], [16], [17], [18], [19], [20]]. The association of ERγ with OA has been confirmed in animal studies using an ER gain-of-function approach [21]. PR is recognized to be more important than the ER in immunomodulation of female prevalence dominant diseases such as systemic lupus erythematosus, rheumatoid arthritis, and OA [6,22,23]. Progressive OA has been reported to sometimes develop during pregnancy and may result from increased hormone levels, including progesterone [24]. Samples of synovial fluid from OA patients demonstrates high levels of cytokines as well as MMPs [[25], [26], [27]]. Progesterone has consistently been found to have inhibitory effects on MMPs in the endometrium and synovial joints [25,27,28], suggesting a connection between the PR and OA. In this study, we aimed to evaluate the role of the PR in subchondral bone. Since both global and bone-cell specific PR knockout mice have higher bone mineral density (BMD) than their wild type (WT) controls and there are observed sex differences in this relationship [5,6,29], we hypothesized that the PR may regulate osteo-chondrocyte metabolism and might have a role in regulating subchondral bone structures, which is related to OA incidence and progression [[30], [31], [32], [33], [34], [35], [36]]. For this reason, we set out to use a Cre recombinase driven approach to examine the effects of tissue-specific inactivation of PR in osteochondral progenitor cells or terminally differentiated chondrocytes on subchondral bone architecture.
Section snippets
Mice
PR-flox mice were obtained from Baylor College of Medicine (Houston, TX, USA). A targeting vector designed to replace part of exon 2 of the PR gene with a selectable marker was employed to create a strain of mice carrying a conditional null PR allele [37]. Prx1-Cre, Col2a1-CreERT(Col2-Cre), Aggrecan-CreERT(Acan-Cre), PR-Cre, mT/mG, and Ai14D transgenic mice were obtained from the Jackson Laboratory (Farmington, CT, USA). The PR-Cre mice (B6.129S(Cg)-Pgrtm1.1(cre)Shah/AndJ) harbor an internal
PR and Col2 expression in bone and joint
To characterize the pattern of PR expression in cartilage, we generated a reporter mouse strain PR-Cre; tdTomato, in which the Cre was driven by the endogenous PR promoter, and this activated the tdTomato expression. We collected the right distal femurs from PR-Cre; tdTomato mice at three weeks of age and created cryosections. Red fluorescence, corresponding to PR expression, was observed within some areas of the growth-plate cartilage as well as the subchondral cartilage area (Fig. 1A, B).
Discussion
Mice lacking PR in osteochondral progenitor cells displayed high subchondral trabecular bone volume and subchondral cortical bone plate thickness, which were associated with higher estimated stiffness and failure load carried by the subchondral bone. The femoral bone formation rate and bone strength were both significantly higher when PR was selectively removed from chondrocytes marked by Col2. Additionally, using Acan-cre [50,53], which was more specific to mature articular chondrocytes, did
Declaration of competing interest
None.
Acknowledgments
SCOR supported these studies: NIH/NIAMS 1P50AR063043 and NIH/NIAMS R01AR061366 (WY). PR-flox mouse was obtained from Dr. John Lydon at Baylor College of Medicine. We thank the SCOR External Advisory Board Member Dr. Mark Johnson, and the Internal Advisory Board Members Dr. Robert Nissenson and Dr. Edward Hsiao for their consultation, technical support, and editorial assistance.
Author contributions
CLD, WY: study conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript. JJJ: collection and assembly of data, data analysis and interpretation, manuscript writing, and final approval of the manuscript. XPL, AK, MJ, LL: collection and assembly of data, data analysis, interpretation, and final approval of the manuscript. BLW: data analysis and interpretation, editing of drafts, and final approval of the
Data and materials availability
Data is archived at the University of California, Davis.
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These authors contributed equally to the study.