Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Nov 1.
Published in final edited form as: J Mol Endocrinol. 2017 Sep 4;59(4):351–363. doi: 10.1530/JME-17-0076

Sexually Dimorphic Regulation of Osteoprogenitor Progesterone in Bone Stromal Cells

Alexander Kot 1, Zhendong Alex Zhong 1,2, Hong Liang Zhang 1,3, Yu-An Evan Lay 1, Nancy E Lane 1, Wei Yao 1
PMCID: PMC5633481  NIHMSID: NIHMS908346  PMID: 28871061

Abstract

Augmenting peak bone mass is a promising strategy to prevent osteoporosis. A mouse model of global progesterone receptor (PR) ablation showed increased bone mass through a sex-dependent mechanism. Cre-Lox recombination was used to generate a mouse model of osteoprogenitor specific PR inactivation, which recapitulated the high bone mass phenotype. In this work, we employed RNA sequencing analysis to evaluate sex-independent and sex-dependent differences in gene transcription of osteoprogenitors of wild type and PR conditional knockout mice. PR deletion caused marked sex hormone-dependent changes in gene transcription in male mice as compared to wild-type controls. These transcriptional differences revealed dysregulation in pathways involving immunomodulation, osteoclasts, bone anabolism, extracellular matrix interaction, and matrix interaction. These results identified many potential mechanisms that may explain our observed high bone mass phenotype with sex differences when PR was selectively deleted in the MSCs.

Keywords: progesterone receptor, osteoprogenitor, signaling pathways, RNA-Seq, bone

Introduction

Osteoporosis is a disease that affects more than 53 million people in the United States (NIH, 2016). Individuals with osteoporosis are usually treated with medications to either maintain or increase bone mass. These treatments include bisphosphonates, parathyroid hormone, RANK ligand inhibitors, and selective estrogen receptor modulators. However, these therapeutic agents are often not prescribed until a patient is diagnosed with osteoporosis. Peak bone mass is associated with the risk of developing osteoporosis in an individual’s lifetime. The greatest gain in bone mass occurs during a rapid bone growth period associated with puberty (Johnston and Slemenda 1993). Bone gain slows and bone mass is maintained through balanced remodeling starting around 25 years of age, lasting for nearly two decades, after which time bone loss begins (Johnston and Slemenda 1993). If a high peak bone mass is achieved, an individual’s risk of osteoporosis would be delayed as the risk of fracture is strongly associated with bone density (Johnston and Slemenda 1993). Attempts to improve peak bone mass through diet and weight-bearing exercise during or before puberty have been somewhat successful, but there is a clear need for interventions with greater potency (Bonjour, et al. 2001; Courteix, et al. 2005; Matkovic, et al. 2005; Ward, et al. 2007).

Progesterone is a sex steroid mostly known for its reproductive system effects. Clinical studies that evaluated the use of progesterone containing oral contraceptives found slight reductions in bone mineral density in the treated group compared to controls at both central and peripheral skeletal locations (Bachrach, et al. 2000; Cromer, et al. 2006; Cundy, et al. 1998; Cundy, et al. 1991; Cundy, et al. 1996; Cundy and Reid 1997; Petitti, et al. 2000). Progesterone’s functions are largely mediated through the progesterone nuclear receptor (PR), a ligand-regulated transcription factor (Conneely and Lydon 2000). The PR is expressed by cultured osteoblasts and osteoclasts (MacNamara and Loughrey 1998; MacNamara, et al. 1995; Pensler, et al. 1990; Wei, et al. 1993; Yao, et al. 2010) and is present in vivo in mouse bone at 4 weeks of age (Zhong, et al. 2015). However, PR’s effect on bone metabolism remains unclear. Our group reported that global PR knockout (PRKO) mice had increased bone mass – female PRKO mice had increased bone formation and male PRKO mice had decreased bone resorption compared to 1–12 month-old wild type (WT) mice (Yao, et al. 2010). Conditional PR inactivation in mesenchymal stem cells (MSCs) as driven by Prx1-Cre (PRcKO) recapitulated the global PRKO phenotype with some differences: 2 month-old male and female mutants had a 85% and 51.5% greater peak bone mass compared to their WT controls, respectively with increased trabecular surface-based bone formation observed in males and decreased osteoclast surface observed in females (Zhong, et al. 2017). Notably, the male Prx1-Cre PRcKO mice had increased MSC frequency in bone marrow stromal cells as well as increased osteogenic potential, as compared to control (Zhong, et al. 2017). Conditional PR inactivation in more terminally differentiated cells using Bglap-Cre and Dmp1-Cre failed to completely reproduce the increased bone phenotype (Zhong, et al. 2017). Therefore, we hypothesized that progesterone signaling through the PR in osteoprogenitors may be a key regulator of sexual dimorphism in the acquisition of peak bone mass.

In this work, we utilized Prx1-Cre to conditionally inactivate the PR gene in MSCs of male and female intact and gonadectomized mice and analyzed differences in gene transcription.

Materials and Methods

Mouse strains and animal procedures

PR-flox B6/129 mice were obtained from Dr. John Lydon at the Baylor College of Medicine. A targeting vector designed to replace part of exon 2 of the PR gene with a selectable marker was used to create a strain of mice carrying a conditional null PR allele (Fernandez-Valdivia, et al. 2010). These PR-flox C57BL/6J mice were then crossed with Prx1-Cre mice (Jackson Laboratory) to create PRcKO mice. The Prx1;PRcKO mouse line were backcrossed at least 10 generations. The efficiency of the Prx1-Cre PRcKO system was validated through undetectable levels of PR in Western blot analysis of femoral protein (Zhong, et al. 2017). One set of PRcKO and WT (Cre-negative, PR-flox/flox) mice were either ovariectomized (OVX) or orchiectomized (ORX) at two months of age and sacrificed at three months of age. Gonadectomy was confirmed by absence of gonads at necropsy, bone mass (microCT), and bone turnover. A second set of PRcKO and WT mice were left intact and sacrificed at 2 months of age. Mice were housed in an animal facility with controlled environmental conditions (12-hour light/dark cycle, temperature 22° C) and had access to food/water ad libitum. PCR-based strategies were used to genotype the mice. At 2 months of age, the mice were exsanguinated by cardiac puncture and dissected. All animal experiments were performed per protocols approved by the University of California, Davis Institutional Animal Care and Use Committee.

Bone marrow-derived osteoprogenitor cells isolation and culture

The condyles of the proximal and distal femurs were removed and the bone marrow was flushed out with phosphate buffered saline. The cell suspension was filtered through a 70μm filter and plated into a dish. The first media change was done less than 72 hours after plating the cells in the flask. These bone marrow stromal cells (BMSCs) were maintained in stem cell medium for 10 days and then were stimulated to differentiate into osteoblasts in osteogenic media containing 50 μg/ml ascorbic acid and 10 mM β-glycerol phosphate for 3 days (Life Technologies, Carlsbad, CA, USA) before being used for RNA isolation and sequencing (n=3–4/genotype/sex).

RNA isolation and sequencing

Transcriptome profiling was performed using a directional, strand-specific mRNA-Seq approach. Briefly, total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA). RNA-Seq libraries were prepared from 0.1 μg total RNA using the KAPA Stranded mRNA-Seq Kit (Kapa Biosystems Inc., Wilmington, MA, USA) per the manufacturer’s standard protocol. Poly-adenylated mRNA was purified from total RNA and ribosomal RNA was removed by binding to oligo(dT) beads, followed by RNA fragmentation at 94ºC in the presence of magnesium. Double-stranded cDNA was generated by random-primed first-strand synthesis and subsequent second strand synthesis in the presence of dUTP for strand marking. The double-stranded cDNA was 3′-A tailed, and indexed, Illumina-compatible adapters were ligated. The libraries were then enriched by high-fidelity PCR amplification (13 cycles) with KAPA HiFi HotStart DNA Polymerase and adapter-specific primers. Subsequently, libraries were be combined for multiplex sequencing on an Illumina HiSeqRNA HiSeq 3000 System (50-bp single read; 8 libraries/lane; ~37 million reads/sample) (Bentley, et al. 2008).

QC and pre-processing

Image processing, base calling, quality scoring (Phred), and sample demultiplexing were executed by HiSeq Control Software with Real Time Analysis (HCS v3.3.41/RTA 2.5.2) and bcl2fastq Conversion Software (Illumina; San Diego, CA). RNA was size-selected, ribosomal RNA depleted, and poly(A)-enriched. Non-coding RNA was not our focus but some, like Xist (polyadenylated ncRNA), were present in our data. Sequence data was analyzed using a standard TopHat-Cufflinks workflow (Trapnell, et al. 2012). RNA-Seq sequence reads were aligned to the reference mouse genome assembly (Dec. 2011, GRCm38/mm10) using TopHat software (Trapnell, et al. 2010), which performs splice junction mapping after read alignment with Bowtie2 (Langmead and Salzberg 2012). Using SubRead (Liao, et al. 2014), aligned reads were summarized using featureCounts and counts-per-million (CPM) values were calculated for each gene. Genes with less than 1 CPM in fewer than 2 samples were removed. Trimmed mean of M-values normalization was applied to each filtered count dataset (Robinson and Oshlack 2010).

Differential expression analysis

Differential gene expression was determined using the edgeR (Robinson, et al. 2010) R/Bioconductor package following the user’s guide examples (Chen Y, et al. 2008). A negative binomial model was fitted to the filtered, normalized data and the trended dispersion was estimated using the Cox-Reid profile-adjusted likelihood. Batch effect was accounted for in generalized linear models and likelihood ratio tests were performed over five comparisons: female WT OVX vs. female PRcKO OVX; male WT ORX vs. male PRcKO ORX; male WT vs. female WT; male WT vs. male PRcKO; and female WT vs. female PRcKO. 2 biological replicates were run for the gonadectomy experiments and 3–4 biological replicates were run for the intact experiments. Differentially expressed genes (DEGs) were identified as genes with −1 > log2(fold change) > 1 and false discovery rates (FDR) < 0.05 by the Benjamini-Hochberg procedure. To reduce the number of identified DEGs and in the male WT vs. male PRcKO comparison, the FDR cutoff was reduced to FDR < 0.01. DEG lists were then analyzed for up- and/or down-regulation enrichment against Kyoto Encyclopedia of Genes and Genome (KEGG) pathways using hypergeometric tests and the Benjamini-Hochberg procedure with FDR < 0.05 (Young, et al. 2010).

qPCR Validation

Differential gene expression was validated in whole bone including bone marrow (n=4–6 biological replicates) using qPCR with primers for Rankl : 5′-CAGCATCGCTCTGTTCCTGTA-3′ (forward) and 5′-CTGCGTTTTCATGGAGTCTCA-3′ (reverse), Opg: 5′-ACCCAGAAACTGGTCATCAGC-3′ (forward) and 5′-CTGCAATACACACACTCATCACT-3′ (reverse), Icam1: 5′-GTGATGCTCAGGTATCCATCCA-3′ (forward) and 5′-CACAGTTCTCAAAGCACAGCG-3′ (reverse), Sdf1: 5′-CTGCACTCACACGACATCAAA-3′ (forward) and 5′-CGCGAATCCGCCAGTAACT-3′ (reverse), Csf1: 5′-ATGAGCAGGAGTATTGCCAAGG-3′ (forward) and 5′-TCCATTCCCAATCATGTGGCTA-3′ (reverse), Trap: 5′-CACTCCCACCCTGAGATTTGT-3′ (forward) and 5′-CATCGTCTGCACGGTTCTG-3′ (reverse), Ccnd1: 5′-GCGTACCCTGACACCAATCTC-3′ (forward) and 5′-CTCCTCTTCGCACTTCTGCTC-3′ (reverse), and Actb: 5′-GGAATGGGTCAGAAGGACTC-3′ (forward) and 5′-CATGTCGTCCCAGTTGGTAA-3′ (reverse). Whole bone RNA was harvested by snap freezing tibiae in liquid nitrogen and homogenizing in Trizol (Bead Ruptor 12, Omni, Inc., Kennesaw, GA, USA; Invitrogen, Carlsbad, CA, USA). Isolated RNA (2 μg/sample) was reverse transcribed to cDNA (RT2 First Strand Kit; SABioscience, Inc., Frederick, MD, USA) and diluted to 10 μL. RT-PCR was carried out on an ABI Prism 7300 (Applied Bioscience, Foster City, CA, USA) in 12.5 μL 2× SYBER Green Mix (SABioscience, Inc., Frederick, MD, USA), 1 μL cDNA, 1 μL primers, and 10.5 μL H2O. PCRs were run in triplicate using 40 cycles of 95 °C, 15s and 60 °C, 60s. Threshold cycle (Ct) was determined for each gene and compared to the reference gene, Act, using the delta-delta Ct method to find log2(fold change). RNA results were plotted against RNA-seq fold change values. Linear regression was performed to assess correlation.

Statistical analysis

Two-tailed Wilcoxon rank-sum tests were used on qPCR data to compare sex-matched WT and PRcKO groups. Statistical significance was determined at p < 0.05. Data were analyzed using the GraphPad Prism 6 software package (La Jolla, CA, USA).

Results

Hormone-dependent effect of PRcKO on MSC transcriptional profiles

We did not detect any significant transcriptome differences in the osteoprogenitor cells in our female WT OVX vs. female PRcKO OVX or male WT ORX vs. male PRcKO ORX contrasts. Hormonal deficiency induced similar changes in bone turnover in WT vs. Prx1;PRcKO mice in both sexes (Figure 1). These results suggested bone turnover and transcriptome changes were hormone-dependent. Further analyses were focused only on the intact WT and PRcKO groups.

Figure 1.

Figure 1

Open in a new tab

Prx1; PRcKO mice and their littermates had similar changes in bone turnover following gonadal deficiency in both sexes. Mice were ovariectomized (OVX) or orchiectomized (ORX) at two-months of age and sacrificed one month post-surgery. Mice were given two fluorescents labeling at −7 and −1 day(s) before sacrifice. (A). Dynamic bone histomorphometry was performed at the distal femurs from wild type and Prx1; PRcKO mice of both sexes at 3 months of age. (B). Representative distal femurs from 2-month-old female and male Prx1; PRcKO mice were OVX or ORX.

DEG identification across comparisons

2796 DEGs were identified in the male WT vs. male PRcKO comparison (851 up-regulated in male PRcKO, 1945 down-regulated in male PRcKO), 2 DEGs in the female WT vs. female PRcKO comparison (2 down-regulated in female PRcKO), and 14 DEGs in the male WT vs. female WT comparison (9 up-regulated in females, 5 down-regulated in females). The DEGs identified in the female WT vs. female PRcKO comparison, Tnxb (log2 FC −1.99, FDR 1.36E-02) and Gldc (log2 FC −2.23, FDR 1.36E-02), were not identified in the male WT vs. male PRcKO comparison.

The male WT vs. female WT sex difference comparison had 14 DEGs. 8 of the DEGs identified in the male WT vs. female WT comparison were also identified as DEGs in the male WT vs. male PRcKO comparison (Table 1).

Table 1.

Wild type sex difference DEGs in common with male wild type vs. male knockout.

Gene Symbol Gene Name log2(FC) between Sexes FDR between Sexes log2(FC) in Males FDR in Males
Xist X-inactive specific transcript −9.71 6.39E-12 3.54 3.81E-03
Mtus2 Microtubule associated tumor suppressor candidate 2 −1.36 1.59E-03 −1.77 2.18E-07
Aldh1a7 Aldehyde dehydrogenase family 1, subfamily A7 −2.57 7.33E-03 3.96 1.07E-06
Tusc5 Tumor suppressor candidate 5 −1.46 8.74E-03 1.31 2.04E-03
Cd300c CMRF35-like molecule 6 5.15 2.66E-02 −7.90 9.92E-10
Aldh1a1 Aldehyde dehydrogenase family 1, subfamily A1 −1.39 2.66E-02 2.45 4.29E-09
Pde3a Phosphodiesterase 3A −1.51 2.66E-02 1.65 3.63E-04
Emx2 Empty spiracles homeobox 2 1.14 3.12E-02 −1.95 3.95E-08
Open in a new tab

Negative fold change values indicate down-regulation in males and down-regulation in the PRcKO group, respectively.

The male WT vs. male PRcKO comparison identified a substantial number of DEGs. We have shown 15 DEGs with the smallest FDR values (Table 2). Because we had evidence supporting many differential gene expressions, we ran KEGG analysis on this comparison to identify biological pathways that may help to explain the increased bone-mass phenotype present in the PRcKO animals including the following pathways (Figure 2).

Table 2.

Top 15 DEGs in male WT vs PRcKO by FDR.

Gene Symbol Gene Name log2(FC) FDR
Msr1 Macrophage scavenger receptor 1 −6.72 8.86E-105
Myo1g Myosin IG −6.94 3.56E-100
Hcls1 Hematopoietic lineage cell-specific Lyn substrate 1 −5.46 1.32E-87
Arhgap30 Rho GTPase activating protein 30 −6.91 1.54E-81
Dok2 Docking protein 2 −5.71 1.85E-81
Hk3 Hexokinase 3 −7.08 4.82E-81
Serping1 Serpin family G member 1 −5.76 9.91E-80
Fmnl1 Formin like 1 −5.21 4.35E-76
Vav1 Vav guanine nucleotide exchange factor 1 −6.68 6.34E-75
Lilrb4a Leukocyte immunoglobulin-like receptor, subfamily B, member 4A −6.28 6.16E-70
Ccr1 C-C chemokine receptor type 1 −5.43 1.26E-68
Inpp5d Inositol polyphosphate-5-phosphatase D −6.45 1.23E-67
Alox5ap Arachidonate 5-lipoxygenase activating protein −7.08 5.30E-66
Fermt3 Fermitin family member 3 −5.66 3.59E-65
Ncf2 Neutrophil cytosolic factor 2 −6.23 8.88E-65
Open in a new tab

Positive fold change values indicate up-regulation in the PRcKO group.

Figure 2.

Figure 2

Open in a new tab

KEGG pathway analysis on DEGs identified in males. (A) Bar plot ranking selected pathways by –log10(FDR) values for enrichment of DEGs up-regulated in PRcKO. (B) Bar plot ranking selected pathways by –log10(FDR) values for enrichment of DEGs down-regulated in PRcKO.

Immunomodulatory pathways in male PRcKO

From KEGG analysis on the male WT vs. male PRcKO comparison DEGs, some of the most significantly overrepresented pathways involved immunomodulation. These pathways included: cytokine-cytokine receptor interaction, chemokine signaling pathway, complement and coagulation cascades, B cell receptor signaling pathway, and T cell receptor signaling pathway (Table 3). Most of the identified DEGs involved in these pathways were down-regulated.

Table 3.

KEGG Pathway analysis results – immunomodulation.

KEGG Pathway Up-regulated genes in PRcKO Down-regulated genes in PRcKO FDR up-regulated FDR down-regulated
Cytokine-cytokine receptor interaction Bmp7, Clcf1, Csf1, Gdf5, Met, Pdgfc, Tnfrsf10b, Tnfrsf11b Ackr3, Acvr1b, Ccl2, Ccl3, Ccl4, Ccl5, Ccl6, Ccl7, Ccl8, Ccl9, Ccl11, Ccl12, Ccl22, Ccr1, Ccr2, Ccr3, Ccr5, Cntfr, Csf1r, Csf2ra, Csf2rb, Csf2rb2, Csf3, Csf3r, Cx3cr1, Cxcl1, Cxcl2, Cxcl5, Cxcl10, Cxcl12, Cxcl14, Cxcl16, Cxcr3, Cxcr4, Egfr, Fas, Flt1, Ifnar2, Il1a, Il1b, Il6, Il1r1, Il1r2, Il1rap, Il2rg, Il3ra, Il4ra, Il6ra, Il7r, Il10ra, Il10rb, Il11ra1, Il13ra1, Il15ra, Il20ra, Il20rb, Il21r, Kit, Lif, Osm, Pdgfb, Pf4, Ppbp, Tnf, Tnfrsf1b, Tnfrsf9, Tnfrsf11a, Tnfrsf13b, Tnfrsf14, Tnfrsf19, Tnfrsf21, Tnfsf8, Tnfsf9, Tnfsf11, Tnfsf13b, Tnfsf14, Vegfa, Vegfc 1.19E-02 4.33E-67
Chemokine signaling Adcy7, Gnb4, Prkcz Adcy5, Arrb2, Ccl2, Ccl3, Ccl4, Ccl5, Ccl6, Ccl7, Ccl8, Ccl9, Ccl11, Ccl12, Ccl22, Ccr1, Ccr2, Ccr3, Ccr5, Cx3cr1, Cxcl1, Cxcl2, Cxcl3, Cxcl5, Cxcl10, Cxcl12, Cxcl16, Cxcr4, Dock2, Fgr, Gnb5, Gng2, Gng4, Gng7, Gngt2, Hck, Jak2, Lyn, Ncf1, Nfkbia, Pf4, Pik3cd, Plcb2, Ppbp, Prex1, Prkcb, Ptk2b, Rac2, Rasgrp2, Shc2, Vav1, Vav3, Was 6.18E-01 3.37E-33
Complement and coagulation cascades Cfd, F2r, Masp1 Bdkrb1, C1qa, C1qb, C1qc, C1ra, C1s1, C2, C3, C3ar1, C4b, C5ar1, C5ar2, Cd55, Cfb, Cfh, F10, F3, F7, Itgam, Itgax, Itgb2, Itgb2l, Kng1, Kng2, Plat, Pros1, Serpind1, Serping1, Tfpi, Vwf 2.41E-02 8.92E-29
B cell receptor signaling N/A Blnk, Btk, Cd22, Cd72, Fcgr2b, Fos, Ifitm1, Inpp5d, Lyn, Nfatc2, Nfkbia, Nfkbie, Pik3ap1, Pik3cd, Pirb, Plcg2, Prkcb, Ptpn6, Rac2, Rac3, Syk, Vav1, Vav3 1.00E+00 5.34E-15
T cell receptor signaling N/A Cd4, Fos, Lcp2, Map3k8, Nfatc2, Nfkbia, Nfkbie, Pik3cd, Prkcq, Ptpn6, Ptprc, Tec, Tnf, Vav1, Vav3 1.00E+00 7.24E-08
Open in a new tab

In the cytokine-cytokine receptor interaction pathway, we found that PRcKO down-regulated CXC chemokines, CXC chemokine receptors, CC chemokines, and CC chemokine receptors. Downstream of the chemokine receptors, PRcKO osteoprogenitors had differential expression in chemokine signaling. Notably, regulatory genes, Jak-STAT signaling genes and actin cytoskeleton regulation genes were all down-regulated.

In the complement cascade, we observed down-regulation of genes in the PRcKO males included the classical and lectin pathways, alternative pathway and their downstream receptors. Three complement cascade genes, Cfd, F2r, and Masp1, were up-regulated in these PRcKO animals. C2 and C4b, central genes in the classical and lectin pathways, were more than 8 fold down-regulated in the PRcKO males. Anaphylatoxin chemotactic receptors C3ar1, C5ar1, and C5ar2 were down-regulated in the PRcKO males. Similarly, we observed down-regulation in many central genes involved in the coagulation cascade.

The B-cell and T-cell receptor signaling pathways had many down-regulated genes in the PRcKO males including those that encode receptors, key signaling molecules and downstream transcription factors.

Osteoclast related pathways in male PRcKO

KEGG analysis revealed down-regulation of genes in pathways related to osteoclasts including: Osteoclast differentiation, NF-kappa B signaling, and TNF signaling (Table 4). In the osteoclast differentiation pathway, three genes were up-regulated in male PRcKO osteoprogenitors: Csf1, Pparg, and Tnfrsf11b. Many receptor genes and their ligands were down-regulated, along with several important signaling genes.

Table 4.

KEGG Pathway analysis results – osteoclast related.

KEGG Pathway Up-regulated genes in PRcKO Down-regulated genes in PRcKO FDR up-regulated FDR down-regulated
Osteoclast differentiation Csf1, Pparg, Tnfrsf11b Acp5, Blnk, Btk, Calcr, Csf1r, Ctsk, Cyba, Fcgr1, Fcgr2b, Fcgr3, Fcgr4, Fos, Gab2, Gm14548, Ifnar2, Il1a, Il1b, Il1r1, Irf9, Itgb3, Junb, Lcp2, Lilra5, Lilra6, Lilrb4a, LOC100038947, Ncf1, Ncf2, Ncf4, Nfatc2, Nfkbia, Oscar, Pik3cd, Pira2, Sirpa, Sirpb1a, Socs3, Spi1, Syk, Tec, Tnf, Tnfrsf11a, Tnfsf11, Trem2, Tyrobp 4.84E-01 1.72E-33
NF-kappa B signaling Bcl2, Bcl2l1, Card10, Pidd1 Bcl2a1b, Birc3, Blnk, Btk, Ccl4, Cd14, Cxcl2, Cxcl12, Icam1, Il1b, Il1r1, Lbp, Lyn, Nfkbia, Plcg2, Prkcb, Prkcq, Syk, Ticam2, Tnf, Tnfaip3, Tnfrsf11a, Tnfsf11, Tnfsf13b, Tnfsf14, Traf1, Vcam1 1.04E-01 5.13E-18
TNF signaling Csf1, Rps6ka4 Bcl3, Birc3, Ccl2, Ccl5, Cebpb, Creb3l1, Creb3l4, Cxcl1, Cxcl2, Cxcl3, Cxcl10, Fas, Fos, Icam1, Ifi47, Il1b, Il6, Junb, Lif, Map3k8, Mmp3, Mmp9, Nfkbia, Nod2, Pik3cd, Socs3, Tnf, Tnfaip3, Tnfrsf1b, Traf1, Vcam1 7.02E-01 1.36E-19
Open in a new tab

Many genes in the NF-kappa B signaling pathway were down-regulated in PRcKO males. Notably, genes in the antigen receptor signaling and genes involved in regulating inflammation downstream of NFkB were down-regulated. Three of the four up-regulated genes, Bcl2, Bcl2l1, and Pidd1 act on cell survival downstream of NFkB.

In the TNF signaling pathway, most of the DEGs were found in genes controlled by TNF signaling including down-regulation in the genes associated with leukocyte recruitment (chemokines), surface receptors (Fas), inflammatory cytokines (Il1b and Il6), intracellular signaling (Bcl3, Ifi47, Socs3, Tnafip3, and Traf1), other transcription factors (Fos), remodeling extracellular matrix (Mmp3, and Mmp9), and cell adhesion (Icam1 and Vcam1). Notably, the Tnf gene was 26-fold down-regulated and the Tnfrsf1b gene was 2.5-fold down-regulated in the PRcKO males.

Bone anabolic pathways in male PRcKO

Next, we selected those pathways which were related to bone anabolism including Wnt signaling and TGF-beta (Table 5). In our male PRcKO osteoprogenitors, we observed multiple genes up-regulated within the canonical Wnt signaling pathway. Genes of inhibitory proteins which were down-regulated were mostly inhibitors for Wnt signaling. Similarly, in the TGF-beta signaling pathway, many central genes were up-regulated in the PRcKO males with inhibitory genes down-regulated.

Table 5.

KEGG Pathway analysis results – bone anabolic.

KEGG Pathway Up-regulated genes in PRcKO Down-regulated genes in PRcKO FDR up-regulated FDR down-regulated
Wnt signaling Cacybp, Ccnd1, Fzd6, Fzd9, Lef1, Peg12, Prkca, Prkcg, Wnt10a, Wnt10b, Wnt11 Camk2a, Camk2b, Nfatc2, Nfatc4, Nkd2, Notum, Plcb2, Porcn, Prkcb, Rac2, Rac3, Sfrp1, Sfrp2, Sfrp4, Wnt2b, Wnt7b, Wnt9a 1.27E-05 1.02E-05
TGF-beta signaling Bmp7, Gdf5, Gdf7, Id1, Id2, Id4, Ltbp1, Rbl1, Smad6, Smad9, Smurf2, Thbs1 Acvr1b, Chrd, Dcn, Pitx2, Tnf 6.78E-09 2.68E-01
Open in a new tab

Extracellular signaling pathways in male PRcKO

Some important pathways involving extracellular signaling were dysregulated in the male PRcKO including PI3K-Akt signaling, MAPK signaling, cGMP-PKG signaling, and cAMP signaling (Table 6). In the PI3K-Akt signaling pathway, genes of many receptors upstream to PI3K and their ligands were dysregulated and many of the pathway’s central genes were down-regulated in the PRcKO males. Notably, some genes encoding proteins phosphorylated by PDK1 were up-regulated in the PRcKO males.

Table 6.

KEGG Pathway analysis results – extracellular signaling.

KEGG Pathway Up-regulated genes in PRcKO Down-regulated genes in PRcKO FDR up-regulated FDR down-regulated
PI3K-Akt signaling Bcl2, Bcl2l1, Brca1, Ccnd1, Ccne2, Chad, Col2a1, Col4a5, Col4a6, Col6a3, Col9a1, Col9a2, Col9a3, Comp, Csf1, Efna5, Epha2, F2r, Fgf2, Fgfr2, Fgfr3, Gnb4, Irs1, Itga2, Itga3, Itga6, Itga10, Itgb7, Lamb3, Met, Pdgfc, Phlpp1, Pkn3, Prkca, Thbs1, Tnn Angpt1, Angpt2, Angpt4, Bcl2l11, Col1a1, Col6a6, Creb3l1, Creb3l4, Csf1r, Csf3, Csf3r, Ddit4, Egfr, Fgf10, Fgf11, Fgf13, Fgf21, Flt1, Gnb5, Gng2, Gng4, Gng7, Gngt2, Gys1, Ifnar2, Igf1, Il2rg, Il3ra, Il4ra, Il6, Il6ra, Il7r, Itga4, Itga7, Itga9, Itgb3, Itgb6, Itgb8, Jak2, Kit, Lama1, Lama4, Lama5, Lamb1, Lpar5, Myb, Nos3, Nr4a1, Osm, Pkc2, Pdgfb, Pik3ap1, Pik3cd, Pik3cg, Pik3r5, Pik3r6, Syk, Thbs3, Tlr2, Vegfa, Vegfc, Vwf 3.82E-23 3.90E-29
MAPK signaling Bdnf, Cacnb2, Dusp5, Fgf2, Fgfr2, Fgfr3, Flnb, Prkca, Prkcg, Rps6ka4, Rps6ka6, Rras2 Arrb2, Cacna1c, Cacna1d, Cacna1f, Cacna1h, Cacna1s, Cacnb1, Cacnb3, Cacng1, Cacng6, Cd14, Dusp2, Dusp3, Egfr, Fas, Fgf10, Fgf11, Fgf13, Fgf21, Fos, Gadd45a, Il1a, Il1b, Il1r1, Il1r2, Map3k8, Map4k1, Mapkapk3, Mef2c, Nr4a1, Ntrk2, Pdgfb, Pla2g4b, Prkcb, Ptpn5, Ptpn7, Rac2, Rac3, Rasgrp2, Rasgrp4, Rps6ka1, Rras, Tnf 1.51E-03 1.82E-19
cGMP-PKG signaling Adcy7, Adra1d, Atp1b1, Irs1, Itpr1, Itpr3, Mylk2, Npr2, Pde3a, Prkg2 Adcy5, Adra2a, Agtr1a, Atp1a2, Atp1a3, Atp1b2, Atp2a1, Atp2a3, Atp2b4, Cacna1c, Cacna1d, Cacna1f, Cacna1s, Creb3l1, Creb3l4, Ednra, Ednrb, Mef2c, Mrvi1, Myh7, Myl9, Mylk, Mylk4, Nfatc2, Nfact4, Nos3, Pde2a, Pde5a, Pik3cg, Pik3r5, Pik3r6, Plcb2 3.91E-04 1.85E-17
cAMP signaling Adcy7, Adcyap1r1, Atp1b1, Bdnf, F2r, Grin3a, Hhip, Pde3a, Ppp1r1b, Rapgef3, Rapgef4, Rras2, Sox9 Adcy5, Arap3, Atp1a2, Atp1a3, Atp1b2, Atp1b4, Cacna1c, Cacna1d, Cacna1f, Cacna1s, Camk2a, Camk2b, Creb3l1, Creb3l4, Ednra, Ffar2, Fos, Fxyd1, Hcar2, Myl9, Nfkbia, Npy, Npy1r, Orai1, Pik3cd, Ptger2, Rac2, Rac3, Rras, Vav1, Vav3, Vipr2 1.41E-06 2.04E-15
Open in a new tab

Matrix interaction pathways in male PRcKO

Finally, we examined pathways involving matrix interaction including: ECM-receptor interaction, focal adhesion, and regulation of actin cytoskeleton (Table 7). We observed dysregulation in ECM components. Many of these ECM-receptor interactions are also involved in the focal adhesion pathway, which was also enriched in both up- and down-regulation. Additionally, some genes of components that associate/bind with integrins were also up- and down-regulated. Some genes involved in regulating focal adhesion were also dysregulated and have been mentioned above in cytokines-cytokine receptor interaction, Wnt signaling, PI3K-Akt signaling and MAPK signaling.

Table 7.

KEGG Pathway analysis results – extracellular matrix interaction.

KEGG Pathway Up-regulated genes in PRcKO Down-regulated genes in PRcKO FDR up-regulated FDR down-regulated
Focal adhesion Actn4, Bcl2, Ccnd1, Chad, Col2a1, Col4a5, Col4a6, Col6a3, Col9a1, Col9a2, Col9a3, Comp, Flnb, Itga2, Itga3, Itga6, Itga10, Itgb7, Lamb3, Met, Mylk2, Pdgfc, Prkca, Prkcg, Thbs1, Tnn Birc3, Cav3, Col1a1, Col6a6, Egfr, Flt1, Igf1, Itga4, Itga7, Itga9, Itgb3, Itgb6, Itgb8, Lama1, Lama4, Lama5, Lamb1, Myl9, Mylk, Mylk4, Mylpf, Parvg, Pdgfb, Pik3cd, Prkcb, Rac2, Rac3, Shc2, Thbs3, Vav1, Vav3, Vegfa, Vegfc, Vwf 4.88E-19 6.60E-14
Regulation of actin cytoskeleton Actn4, Diap3, F2r, Fgd1, Fgf2, Fgfr2, Fgfr3, Iqgap3, Itga2, Itga3, Itga6, Itga10, Itgb7, Myh10, Mylk2, Pdgfc, Pfn2, Rras2 Bdkrb1, Cd14, Cyfip2, Egfr, Fgf10, Fgf11, Fgf13, Fgf21, Itga4, Itga7, Itga9, Itgal, Itgam, Itgax, Itgb2, Itgb2l, Itgb3, Itgb6, Itgb8, Myl9, Mylk, Mylk4, Mylpf, Nckap1l, Pdgfb, Pik3cd, Pip5k1b, Rac2, Rac3, Rras, Scin, Vav1, Vav3, Was 4.87E-09 1.01E-13
ECM-receptor interaction Chad, Col2a1, Col4a5, Col4a6, Col6a3, Col9a1, Col9a2, Col9a3, Comp, Hmmr, Itga2, Itga3, Itga6, Itga10, Itgb7, Lamb3, Thbs1, Tnn Cd36, Col1a1, Col6a6, Itga4, Itga7, Itga9, Itgb3, Itgb6, Itgb8, Lama1, Lama4, Lama5, Lamb1, Npnt, Sdc1, Thbs3, Vwf 5.04E-20 2.92E-09
Open in a new tab

qPCR expression of select genes

The RNA-seq results were validated using qPCR on the same RNA samples. Linear regression on the qPCR vs. RNA log2 (fold change) values indicated good correlation (y = 0.9203× −0.6699; R2 = 0.8916; p<0.0001; Figure 3A). In the osteoprogenitor RNA, the Rankl/Opg ratio was decreased in the PRcKOs (9.270 in male WT vs. −37.037 in male PRcKO; 6.131 in female WT vs. 3.006 in female PRcKO). qPCR was also used to examine transcription of select genes in the entire bone. Whole bone responded similarly to PRcKO as osteoprogenitors – most genes were down-regulated and males experience a greater effect (Figure 3B). Rankl, Opg, Trap, and Ccnd1 were all significantly down-regulated in the male PRcKOs. None of the genes examined were significantly dysregulated in the female PRcKOs.

Figure 3.

Figure 3

Open in a new tab

Validation of select gene expressions by qRT-PCR. (A) qPCR vs RNA-seq for selected genes. log2 (fold change) values plotted and used to assess correlation. (B) Whole bone RT-PCR for selected genes (n=4–6/group). Mean +/− SEM (* p < 0.05; Mann-Whitney test vs. WT).

Discussion

Compared to other sex steroid receptors such as the estrogen and androgen-receptors, even less is known about the progesterone receptor’s (PR) effect on bone cell metabolism. The PR has been shown to be important for sex differences in bone mass acquisition, as genetic and chemical strategies to inhibit PR signaling have been shown to increase peak bone mass accrual, with sex differences (Yao, et al. 2010). Previous studies with the Prx1-Cre driven PRcKO system have demonstrated efficient PR inactivation by Western blot analysis, a sex-dependent increase in MSC frequency in bone marrow stromal cells, and greater osteogenic potential of those MSCs (Zhong, et al. 2017). This current report revealed a critical role of PR in MSCs and that the lack of PR signaling in the MSCs significantly affects pathways involving immunomodulation, bone remodeling, extracellular signaling, and matrix interactions. Most importantly, these effects were only seen in the males and were hormone (ligand)-dependent. These finding suggest that the PR signaling in MSCs intrinsically regulates sex differences in health and disease conditions.

In cultured osteoprogenitors, the male WT vs. male PRcKO comparison had many more DEGs compared to the other comparisons (male WT vs. female WT and female WT vs. female PRcKO). Further analysis identified many KEGG pathways as enriched for DEGs in this male WT vs. male PRcKO comparison. In line with our group’s reported bone mass findings (Zhong, et al. 2017), PRcKO had a greater effect on male gene transcription compared to females.

The effects of progesterone and PR on immunomodulation are well described for pregnancy and regulation of menstrual cycles. Progesterone, mediated through PR, decreased bone marrow derived dendritic cells and the effects were more pronounced in the females than the males (Butts, et al. 2008). The presence of progesterone and its interaction with progesterone receptors at the decidua level appears to play a major role in the modulation of maternal immune response and suppression of inflammation (Di Renzo, et al. 2016). Blocking PR was associated with high NK activity as well as induction of pro-inflammatory cytokines TNFα, IFN-γ and NF-κB that increased resorption rate (Szekeres-Bartho, et al. 2005; Tiwari, et al. 2016). In the nervous system, after a spinal cord injury, PR modulates the astrocyte response and commitment of oligodendrocyte progenitor cells to enhance remyelination (Labombarda, et al. 2015) and progesterone treatment decreases neuropathic pain associated with spinal cord injury (Coronel, et al. 2016). The absence of PR signaling in the MSCs exerted an immunosuppressive response that was more pronounced in the males, suggesting that PR expression levels may elucidate disparities in gender specific immune disorders and the potential role for targeting PR as gender-specific medicine.

In the female reproductive tract and brain, the progesterone receptor is reported to regulate complement components, particularly C3 (Brinton, et al. 2008; Hasty, et al. 1994; Lundeen et al. 2001). Anaphylatoxins, C3a and C5a, have been reported to be chemotactic factors for MSCs and osteoblasts, with stronger effects in osteoblasts which may be due to increased C5aR expression (Schraufstatter, et al. 2009; Ignatius, et al. 2009; Schoengraf, et al. 2013). C3 expression is increased by osteoclastogenesis and C3aR signaling has been shown to stimulate osteoblast differentiation (Matsuoka, et. al 2014). Inactivating the PR in male MSCs decreased the mRNA for both C3aR and C5aR, as well as many other complement-related genes which may signal resistance to complement.

Apart from the classical progesterone nuclear receptor, membrane associated non-genomic binding protein for progesterone have been cloned. These membrane-binding sites for progesterone have been described as the membrane-associated binding protein and the mPRs (Kazeto, et al. 2005). The membrane associated progesterone-binding proteins are termed 25-Dx, progesterone receptor membrane component 1 (PGRMC 1) and Hpr. 6 (Krebs, et al. 2000; Meyer, et al. 1996; Peluso, et al. 2006; Selmin, et al. 2005). These proteins are associated with progesterone’s nongenomic actions including the activation of signal transduction pathways that stimulate the intracellular MAPK signaling (Duckworth, et al. 2002; Hammes 2003; Krietsch, et al. 2006; Maller 2001). Activation of the MAPK pathway is the key extranuclear signaling action of the steroid’s regulation of cell proliferation and survival in various cell types including osteoblasts and osteocytes (Martin 2001; Marzia, et al. 2000; Plotkin, et al. 1999). The MAPK pathway promotes osteoblast survival and has been shown to contribute to the anabolic effects of parathyroid hormone (Aghaloo, et al. 2006; Kwok, et al. 2005; Qin, et al. 2005), basic fibroblast growth factor (Jackson, et al. 2007) and lactoferrin (Cornish, et al. 2006; Grey, et al. 2006; Naot, et al. 2005). PGRMC1 may serve as an adaptor protein to regulate immunoreceptor tyrosine-based activation motif/ITAM signaling, which is known for its roles in vesicle targeting and osteoclast signaling (Fodor, et al. 2006; Humphrey, et al. 2005; Shinohara and Takayanagi 2007; Takayanagi 2005). However, the net effect of MAPK signaling in osteoprogenitors is currently unclear (Schindeler and Little 2006).

The effects of PR inactivation may have been amplified in males and/or offset in females. The biosynthesis of the sex hormones is very closely related and it is challenging to study effects of individual hormones (Heffner, et al. 2010). It is possible that other hormones played a role in creating the sexual dimorphism in bone mass and gene transcription phenotypes. Additionally, the PR shares the same DNA response element with the receptors for androgens, glucocorticoids, and mineralcortocoids (Beato, et al. 1995). Inactivating PR signaling may have allowed other nuclear receptors to access the shared response elements.

This study was comprehensive follow-up to our previous findings that global PR KO mice had increased bone mass acquisition compared to their wild-type littermates, with sex differences and identified those genes that may control this observation. However, there were some limitations to this study. We used a single Cre recombinase to inactivate PR signaling and analyzed RNA from a single cell-type, which limited our findings. Because of our osteoprogenitor isolation and culture technique, we expected some levels of bone marrow contamination, which may have confounded some of our results. Due to the relatively small sample sizes, we were not powered to find lower differential gene expressions, particularly in our female experiment. Also, we were not able to differentiate between PR’s direct targets and indirect changes. These limitations are largely inherent to the techniques that we used and can be mitigated through additional investigations by using Cre recombinases that target different cell populations.

Overall, these findings have identified many potential mechanisms that may explain our observed high bone mass phenotype with sex differences when PR was selectively deleted in the MSCs. Additional functional studies on progesterone receptor signaling in both osteoprogenitors and cells later in the osteoblast lineage are necessary to better understand the context of PR signaling in the regulation of sexual dimorphism in health and disease conditions.

Acknowledgments

Funding

This work was supported by the NIH/NIAMS (SCOR: 1P50AR063043 and R01AR061366).

The authors are grateful to Ryan R. Davis for his expert technical assistance with Next-Generation Sequencing.

Footnotes

Declaration of Interest

The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.

References

  1. Aghaloo TL, Pirih FQ, Shi A, Bezouglaia O, Tetradis S. Parathyroid hormone induces mitogen-activated kinase phosphatase 1 in murine osteoblasts primarily through cAMP-protein kinase A signaling. J Periodontol. 2006;77:21–30. doi: 10.1902/jop.2006.77.1.21. [DOI] [PubMed] [Google Scholar]
  2. Bachrach LK, Cundy T, Ott SM. Depot medroxyprogesterone acetate in teens: A risk for bone health? Pediatrics. 2000;106:1137–1138. doi: 10.1542/peds.106.5.1137. [DOI] [PubMed] [Google Scholar]
  3. Beato M, Herrlich P, Schütz G. Steroid Hormone Receptors: Many Actors in Search of a Plot. Cell. 1995;83:851–857. doi: 10.1016/0092-8674(95)90201-5. [DOI] [PubMed] [Google Scholar]
  4. Bentley DR, Balasubramanian S, Swerdlow HP, Smith GP, Milton J, Brown CG, Hall KP, Evers DJ, Barnes CL, Bignell HR, et al. Accurate whole human genome sequencing using reversible terminator chemistry. Nature. 2008;456:53–59. doi: 10.1038/nature07517. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bonjour JP, Chevalley T, Ammann P, Slosman D, Rizzoli R. Gain in bone mineral mass in prepubertal girls 3.5 years after discontinuation of calcium supplementation: a follow-up study. Lancet. 2001;358:1208–1212. doi: 10.1016/S0140-6736(01)06342-5. [DOI] [PubMed] [Google Scholar]
  6. Brinton RD, Thompson RF, Foy MR, Baudry M, Wang JM, Finch CE, Morgan TE, Pike CJ, Mack WJ, Stanczyk FZ, Nilsen J. Progesterone receptors: Form and function in brain. Front Neuroendocrinol. 2008;29:313–339. doi: 10.1016/j.yfrne.2008.02.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Butts CL, Bowers E, Horn JC, Shukair SA, Belyavskaya E, Tonelli L, Sternberg EM. Inhibitory effects of progesterone differ in dendritic cells from female and male rodents. Gend Med. 2008;5:434–447. doi: 10.1016/j.genm.2008.11.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chen YMD, Ritchie M, Robinson M, Smyth GK. edgeR: differential expression analysis of digital gene expression data. Bioconductor. 2008 doi: 10.1093/bioinformatics/btp616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Conneely OM, Lydon JP. Progesterone receptors in reproduction: functional impact of the A and B isoforms. Steroids. 2000;65:571–577. doi: 10.1016/s0039-128x(00)00115-x. [DOI] [PubMed] [Google Scholar]
  10. Cornish J, Palmano K, Callon KE, Watson M, Lin JM, Valenti P, Naot D, Grey AB, Reid IR. Lactoferrin and bone; structure-activity relationships. Biochem Cell Biol. 2006;84:297–302. doi: 10.1139/o06-057. [DOI] [PubMed] [Google Scholar]
  11. Coronel MF, Raggio MC, Adler NS, De Nicola AF, Labombarda F, Gonzalez SL. Progesterone modulates pro-inflammatory cytokine expression profile after spinal cord injury: Implications for neuropathic pain. J Neuroimmunol. 2016;292:85–92. doi: 10.1016/j.jneuroim.2016.01.011. [DOI] [PubMed] [Google Scholar]
  12. Courteix D, Jaffre C, Lespessailles E, Benhamou L. Cumulative effects of calcium supplementation and physical activity on bone accretion in premenarchal children: a double-blind randomised placebo-controlled trial. Int J Sports Med. 2005;26:332–338. doi: 10.1055/s-2004-821040. [DOI] [PubMed] [Google Scholar]
  13. Cromer BA, Scholes D, Berenson A, Cundy T, Clark MK, Kaunitz AM Society for Adolescent M. Depot medroxyprogesterone acetate and bone mineral density in adolescents--the Black Box Warning: a Position Paper of the Society for Adolescent Medicine. J Adolesc Health. 2006;39:296–301. doi: 10.1016/j.jadohealth.2006.03.011. [DOI] [PubMed] [Google Scholar]
  14. Cundy T, Cornish J, Roberts H, Elder H, Reid IR. Spinal bone density in women using depot medroxyprogesterone contraception. Obstet Gynecol. 1998;92:569–573. doi: 10.1016/s0029-7844(98)00270-1. [DOI] [PubMed] [Google Scholar]
  15. Cundy T, Evans M, Roberts H, Wattie D, Ames R, Reid IR. Bone density in women receiving depot medroxyprogesterone acetate for contraception. BMJ. 1991;303:13–16. doi: 10.1136/bmj.303.6793.13. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cundy T, Farquhar CM, Cornish J, Reid IR. Short-term effects of high dose oral medroxyprogesterone acetate on bone density in premenopausal women. J Clin Endocrinol Metab. 1996;81:1014–1017. doi: 10.1210/jcem.81.3.8772567. [DOI] [PubMed] [Google Scholar]
  17. Cundy T, Reid IR. Bone loss and depot medroxyprogesterone. Am J Obstet Gynecol. 1997;176:1116–1117. doi: 10.1016/s0002-9378(97)70415-0. [DOI] [PubMed] [Google Scholar]
  18. Di Renzo GC, Giardina I, Clerici G, Brillo E, Gerli S. Progesterone in normal and pathological pregnancy. Horm Mol Biol Clin Investig. 2016;27:35–48. doi: 10.1515/hmbci-2016-0038. [DOI] [PubMed] [Google Scholar]
  19. Duckworth BC, Weaver JS, Ruderman JV. G2 arrest in Xenopus oocytes depends on phosphorylation of cdc25 by protein kinase A. Proc Natl Acad Sci U S A. 2002;99:16794–16799. doi: 10.1073/pnas.222661299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Fernandez-Valdivia R, Jeong J, Mukherjee A, Soyal SM, Li J, Ying Y, Demayo FJ, Lydon JP. A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland. Genesis. 2010;48:106–113. doi: 10.1002/dvg.20586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Fodor S, Jakus Z, Mocsai A. ITAM-based signaling beyond the adaptive immune response. Immunol Lett. 2006;104:29–37. doi: 10.1016/j.imlet.2005.11.001. [DOI] [PubMed] [Google Scholar]
  22. Grey A, Zhu Q, Watson M, Callon K, Cornish J. Lactoferrin potently inhibits osteoblast apoptosis, via an LRP1-independent pathway. Mol Cell Endocrinol. 2006;251:96–102. doi: 10.1016/j.mce.2006.03.002. [DOI] [PubMed] [Google Scholar]
  23. Hammes SR. The further redefining of steroid-mediated signaling. Proc Natl Acad Sci U S A. 2003;100:2168–2170. doi: 10.1073/pnas.0530224100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Hasty LA, Lambris JD, Lessey BA, Pruksananonda K, Lyttle CR. Hormonal regulation of complement components and receptors throughout the menstral cycle. Am J Obstet Gynecol. 1994;170:168–175. doi: 10.1016/s0002-9378(94)70404-x. [DOI] [PubMed] [Google Scholar]
  25. Heffner LJ, Schust DJ. The Reproductive System at a Glance. 3. West Sussex, UK: John Wiley and Sons; 2010. p. 16. [Google Scholar]
  26. Humphrey MB, Lanier LL, Nakamura MC. Role of ITAM-containing adapter proteins and their receptors in the immune system and bone. Immunol Rev. 2005;208:50–65. doi: 10.1111/j.0105-2896.2005.00325.x. [DOI] [PubMed] [Google Scholar]
  27. Ignatius A, Ehrnthaller C, Brenner RE, Kreja L, Schoengraf P, Lisson P, Blakytny R, Recknagel S, Claes L, Gebhard F, Lambris JD, Huber-Lang M. The anaphylatoxin receptor C5aR is present during fracture healing in rats and mediates osteoblast migration in vitro. J Trauma. 2011;71:952–960. doi: 10.1097/TA.0b013e3181f8aa2d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jackson RA, Murali S, van Wijnen AJ, Stein GS, Nurcombe V, Cool SM. Heparan sulfate regulates the anabolic activity of MC3T3-E1 preosteoblast cells by induction of Runx2. J Cell Physiol. 2007;210:38–50. doi: 10.1002/jcp.20813. [DOI] [PubMed] [Google Scholar]
  29. Johnston CC, Jr, Slemenda CW. Determinants of peak bone mass. Osteoporos Int. 1993;3(Suppl 1):54–55. doi: 10.1007/BF01621864. [DOI] [PubMed] [Google Scholar]
  30. Kazeto Y, Goto-Kazeto R, Thomas P, Trant JM. Molecular characterization of three forms of putative membrane-bound progestin receptors and their tissue-distribution in channel catfish, Ictalurus punctatus. J Mol Endocrinol. 2005;34:781–791. doi: 10.1677/jme.1.01721. [DOI] [PubMed] [Google Scholar]
  31. Krebs CJ, Jarvis ED, Chan J, Lydon JP, Ogawa S, Pfaff DW. A membrane-associated progesterone-binding protein, 25-Dx, is regulated by progesterone in brain regions involved in female reproductive behaviors. Proc Natl Acad Sci U S A. 2000;97:12816–12821. doi: 10.1073/pnas.97.23.12816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Krietsch T, Fernandes MS, Kero J, Losel R, Heyens M, Lam EW, Huhtaniemi I, Brosens JJ, Gellersen B. Human homologs of the putative G protein-coupled membrane progestin receptors (mPRalpha, beta, and gamma) localize to the endoplasmic reticulum and are not activated by progesterone. Mol Endocrinol. 2006;20:3146–3164. doi: 10.1210/me.2006-0129. [DOI] [PubMed] [Google Scholar]
  33. Kwok S, Qin L, Partridge NC, Selvamurugan N. Parathyroid hormone stimulation and PKA signaling of latent transforming growth factor-beta binding protein-1 (LTBP-1) mRNA expression in osteoblastic cells. J Cell Biochem. 2005;95:1002–1011. doi: 10.1002/jcb.20453. [DOI] [PubMed] [Google Scholar]
  34. Labombarda F, Jure I, Gonzalez S, Lima A, Roig P, Guennoun R, Schumacher M, De Nicola AF. A functional progesterone receptor is required for immunomodulation, reduction of reactive gliosis and survival of oligodendrocyte precursors in the injured spinal cord. J Steroid Biochem Mol Biol. 2015;154:274–284. doi: 10.1016/j.jsbmb.2015.09.011. [DOI] [PubMed] [Google Scholar]
  35. Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–359. doi: 10.1038/nmeth.1923. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics. 2014;30:923–930. doi: 10.1093/bioinformatics/btt656. [DOI] [PubMed] [Google Scholar]
  37. Lundeen SG, Zhang Z, Zhu Y, Carver JM, Winneker RC. Rat uterine complement C3 expression as a model for progesterone receptor modulators: characterization of the new progestin trimegestone. J Steroid Biochem Mol Biol. 2001;28:137–143. doi: 10.1016/s0960-0760(01)00091-7. [DOI] [PubMed] [Google Scholar]
  38. MacNamara P, Loughrey HC. Messenger RNA transcripts coding for progesterone receptor A and B isoforms are expressed in human osteoblast cells. Biochem Soc Trans. 1998;26:1–7. doi: 10.1042/bst0260001. [DOI] [PubMed] [Google Scholar]
  39. MacNamara P, O’Shaughnessy C, Manduca P, Loughrey HC. Progesterone receptors are expressed in human osteoblast-like cell lines and in primary human osteoblast cultures. Calcif Tissue Int. 1995;57:436–441. doi: 10.1007/BF00301947. [DOI] [PubMed] [Google Scholar]
  40. Maller JL. The elusive progesterone receptor in Xenopus oocytes. Proc Natl Acad Sci U S A. 2001;98:8–10. doi: 10.1073/pnas.98.1.8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Martin GS. The hunting of the Src. Nat Rev Mol Cell Biol. 2001;2:467–475. doi: 10.1038/35073094. [DOI] [PubMed] [Google Scholar]
  42. Marzia M, Sims NA, Voit S, Migliaccio S, Taranta A, Bernardini S, Faraggiana T, Yoneda T, Mundy GR, Boyce BF, et al. Decreased c-Src expression enhances osteoblast differentiation and bone formation. J Cell Biol. 2000;151:311–320. doi: 10.1083/jcb.151.2.311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Matkovic V, Goel PK, Badenhop-Stevens NE, Landoll JD, Li B, Ilich JZ, Skugor M, Nagode LA, Mobley SL, Ha EJ, et al. Calcium supplementation and bone mineral density in females from childhood to young adulthood: a randomized controlled trial. Am J Clin Nutr. 2005;81:175–188. doi: 10.1093/ajcn/81.1.175. [DOI] [PubMed] [Google Scholar]
  44. Matsuoka K, Park K, Ito M, Ikeda K, Takeshita S. Osteoclast-Derived Complement Component 3a Stimulates Osteoblast Differentiation. J Bone Miner Res. 2014;29:1522–1530. doi: 10.1002/jbmr.2187. [DOI] [PubMed] [Google Scholar]
  45. Meyer C, Schmid R, Scriba PC, Wehling M. Purification and partial sequencing of high-affinity progesterone-binding site(s) from porcine liver membranes. Eur J Biochem. 1996;239:726–731. doi: 10.1111/j.1432-1033.1996.0726u.x. [DOI] [PubMed] [Google Scholar]
  46. Naot D, Grey A, Reid IR, Cornish J. Lactoferrin--a novel bone growth factor. Clin Med Res. 2005;3:93–101. doi: 10.3121/cmr.3.2.93. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. NIH Handout on Health: Osteoporosis. 2016;16–5158:1–44. [Google Scholar]
  48. Peluso JJ, Pappalardo A, Losel R, Wehling M. Progesterone membrane receptor component 1 expression in the immature rat ovary and its role in mediating progesterone’s antiapoptotic action. Endocrinology. 2006;147:3133–3140. doi: 10.1210/en.2006-0114. [DOI] [PubMed] [Google Scholar]
  49. Pensler JM, Radosevich JA, Higbee R, Langman CB. Osteoclasts isolated from membranous bone in children exhibit nuclear estrogen and progesterone receptors. J Bone Miner Res. 1990;5:797–802. doi: 10.1002/jbmr.5650050802. [DOI] [PubMed] [Google Scholar]
  50. Petitti DB, Piaggio G, Mehta S, Cravioto MC, Meirik O. Steroid hormone contraception and bone mineral density: a cross-sectional study in an international population. The WHO Study of Hormonal Contraception and Bone Health. Obstet Gynecol. 2000;95:736–744. doi: 10.1016/s0029-7844(00)00782-1. [DOI] [PubMed] [Google Scholar]
  51. Plotkin LI, Weinstein RS, Parfitt AM, Roberson PK, Manolagas SC, Bellido T. Prevention of osteocyte and osteoblast apoptosis by bisphosphonates and calcitonin. J Clin Invest. 1999;104:1363–1374. doi: 10.1172/JCI6800. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Qin L, Li X, Ko JK, Partridge NC. Parathyroid hormone uses multiple mechanisms to arrest the cell cycle progression of osteoblastic cells from G1 to S phase. J Biol Chem. 2005;280:3104–3111. doi: 10.1074/jbc.M409846200. [DOI] [PubMed] [Google Scholar]
  53. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics. 2010;26:139–140. doi: 10.1093/bioinformatics/btp616. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Robinson MD, Oshlack A. A scaling normalization method for differential expression analysis of RNA-seq data. Genome Biol. 2010;11:R25. doi: 10.1186/gb-2010-11-3-r25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Schindeler A, Little DG. Ras-MAPK signaling in osteogenic differentiation: friend or foe? J Bone Miner Res. 2006;21:1331–1338. doi: 10.1359/jbmr.060603. [DOI] [PubMed] [Google Scholar]
  56. Schoengraf P, Lambris JD, Recknagel S, Kreja L, Liedert A, Brenner RE, Huber-Lang M, Ignatius A. Does complement play a role in bone development and regeneration? Immunobiology. 2013;218:1–9. doi: 10.1016/j.imbio.2012.01.020. [DOI] [PubMed] [Google Scholar]
  57. Schraufstatter IU, Discipio RG, Zhao M, Khaldoyanidi SK. C3a and C5a are chemotactic factors for human mesenchymal stem cells, which cause prolonged ERK1/2 phosphorylation. J Immunol. 2009;182:3827–3836. doi: 10.4049/jimmunol.0803055. [DOI] [PubMed] [Google Scholar]
  58. Selmin O, Thorne PA, Blachere FM, Johnson PD, Romagnolo DF. Transcriptional activation of the membrane-bound progesterone receptor (mPR) by dioxin, in endocrine-responsive tissues. Mol Reprod Dev. 2005;70:166–174. doi: 10.1002/mrd.20090. [DOI] [PubMed] [Google Scholar]
  59. Shinohara M, Takayanagi H. Novel osteoclast signaling mechanisms. Curr Osteoporos Rep. 2007;5:67–72. doi: 10.1007/s11914-007-0005-1. [DOI] [PubMed] [Google Scholar]
  60. Szekeres-Bartho J, Polgar B, Kozma N, Miko E, Par G, Szereday L, Barakonyi A, Palkovics T, Papp O, Varga P. 2005 Progesterone-dependent immunomodulation. In: Markert UR, editor. Immunology of pregnancy. Basel (Switzerland): Karger; [DOI] [PubMed] [Google Scholar]
  61. Takayanagi H. Mechanistic insight into osteoclast differentiation in osteoimmunology. J Mol Med. 2005;83:170–179. doi: 10.1007/s00109-004-0612-6. [DOI] [PubMed] [Google Scholar]
  62. Tiwari D, Bose PD, Sultana R, Das CR, Bose S. Preterm delivery and associated negative pregnancy outcome - A tale of faulty progesterone receptor signalling pathway and linked derailed immunomodulation: A study from Northeast India. J Reprod Immunol. 2016;118:76–84. doi: 10.1016/j.jri.2016.10.001. [DOI] [PubMed] [Google Scholar]
  63. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L. Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012;7:562–578. doi: 10.1038/nprot.2012.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010;28:511–515. doi: 10.1038/nbt.1621. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Ward KA, Roberts SA, Adams JE, Lanham-New S, Mughal MZ. Calcium supplementation and weight bearing physical activity--do they have a combined effect on the bone density of pre-pubertal children? Bone. 2007;41:496–504. doi: 10.1016/j.bone.2007.06.007. [DOI] [PubMed] [Google Scholar]
  66. Wei LL, Leach MW, Miner RS, Demers LM. Evidence for progesterone receptors in human osteoblast-like cells. Biochem Biophys Res Commun. 1993;195:525–532. doi: 10.1006/bbrc.1993.2077. [DOI] [PubMed] [Google Scholar]
  67. Yao W, Dai W, Shahnazari M, Pham A, Chen Z, Chen H, Guan M, Lane NE. Inhibition of the progesterone nuclear receptor during the bone linear growth phase increases peak bone mass in female mice. PLoS One. 2010;5:e11410. doi: 10.1371/journal.pone.0011410. [DOI] [PMC free article] [PubMed] [Google Scholar]
  68. Young MD, Wakefield MJ, Smyth GK, Oshlack A. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010;11:R14. doi: 10.1186/gb-2010-11-2-r14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Zhong ZA, Kot A, Lay YE, Zhang H, Jia J, Lane NE, Yao W. Sex-Dependent, Osteoblast Stage-Specific Effects of Progesterone Receptor on Bone Acquisition. J Bone Miner Res. 2017 doi: 10.1002/jbmr.3186. In Press. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Zhong ZA, Sun W, Chen H, Zhang H, Lane NE, Yao W. Inactivation of the Progesterone Receptor in Mx1+ Cells Potentiates Osteogenesis in Calvaria but Not in Long Bone. PLoS One. 2015;10:e0139490. doi: 10.1371/journal.pone.0139490. [DOI] [PMC free article] [PubMed] [Google Scholar]

RESOURCES