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Endometriosis is a gynecological disease prevalent in women of reproductive age, and it is characterized by the ectopic presence and growth of the eutopic endometrium. The pathophysiology and diagnostic biomarkers of endometriosis have not yet been comprehensively determined. To discover molecular markers and pathways underlying the pathogenesis of endometriosis, we identified differentially expressed genes (DEGs) in three Gene Expression Omnibus microarray datasets (GSE11691, GSE23339, and GSE7305) and performed gene set enrichment analysis (GSEA) and protein–protein interaction (PPI) network analyses. We also validated the identified genes via immunohistochemical analysis of tissues obtained from patients with endometriosis or healthy volunteers. A total of 118 DEGs (79 upregulated and 39 downregulated) were detected in each dataset with a lower (fold change) FC cutoff (log2|FC| > 1), and 17 DEGs (11 upregulated and six downregulated) with a higher FC cutoff (log2|FC| > 2). KEGG and GO functional analyses revealed enrichment of signaling pathways associated with inflammation, complement activation, cell adhesion, and extracellular matrix in endometriotic tissues. Upregulation of seven genes (C7, CFH, FZD7, LY96, PDLIM3, PTGIS, and WISP2) out of 17 was validated via comparison with external gene sets, and protein expression of four genes (LY96, PDLIM3, PTGIS, and WISP2) was further analyzed by immunohistochemistry and western blot analysis. Based on these results, we suggest that TLR4/NF-κB and Wnt/frizzled signaling pathways, as well as estrogen receptors, regulate the progression of endometriosis. These pathways may be therapeutic and diagnostic targets for endometriosis.

The essential osteoblast-related transcription factor Runx2 and the female steroid hormone estrogen are known to play pivotal roles in bone homeostasis; however, the functional interaction between Runx2- and estrogen-mediated signaling in skeletal tissues is minimally understood. Here we provide evidence that aromatase (CYP19), a rate-limiting enzyme responsible for estrogen biosynthesis in mammals, is transcriptionally regulated by Runx2. Consistent with the presence of multiple Runx2 binding sites, the binding of Runx2 to the aromatase promoter was demonstrated in vitro and confirmed in vivo by chromatin immunoprecipitation assays. The bone-specific aromatase promoter is activated by Runx2, and endogenous aromatase gene expression is upregulated by Runx2 overexpression, establishing the aromatase gene as a target of Runx2. The biological significance of the Runx2 transcriptional control of the aromatase gene is reflected by the enhanced estrogen biosynthesis in response to Runx2 in cultured cells. Reduced in vivo expression of skeletal aromatase gene and low bone mineral density are evident in Runx2 mutant mice. Collectively, these findings uncover a novel link between Runx2-mediated osteoblastogenic processes and the osteoblast-mediated biosynthesis of estrogen as an osteoprotective steroid hormone.

The transdifferentiation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells has been implicated in the context of vascular calcification. We investigated the roles of vitamin D receptor (Vdr) and runt-related transcription factor 2 (Runx2) in the osteoblastic differentiation of VSMCs in response to vitamin D-3 using in vitro VSMCs cultures and in vivo in Vdr knockout (Vdr(-/-)) and Runx2 carboxy-terminus truncated heterozygous (Runx2(+/Delta C)) mice. Treatment of VSMCs with active vitamin D-3 promoted matrix mineral deposition, and increased the expressions of Vdr, Runx2, and of osteoblastic genes but decreased the expression of smooth muscle myosin heavy chain in primary VSMCs cultures. Immunoprecipitation experiments suggested an interaction between Vdr and Runx2. Furthermore, silencing Vdr or Runx2 attenuated the procalcific effects of vitamin D-3. Functional cooperation between Vdr and Runx2 in vascular calcification was also confirmed in in vivo mouse models. Vascular calcification induced by high-dose vitamin D-3 was completely inhibited in Vdr(-/-) or Runx2(+/Delta C) mice, despite elevated levels of serum calcium or alkaline phosphatase. Collectively, these findings suggest that functional cooperation between Vdr and Runx2 is necessary for vascular calcification in response to vitamin D-3.

Vascular calcification refers to the deposition of calcium phosphate in cardiovascular tissues, including arteries and myocardium. Vascular calcification is frequently associated with cardiovascular disease. Recently, bone morphgenetic protein-7 (BMP-7) has been proposed to play an inhibitory role in vascular calcification, but its inhibitory effect has not been fully elucidated. We therefore tested the hypothesis that BMP-7 inhibits vascular calcification by using two conditions, high levels of vitamin D and phosphate, each of which could enhance vascular calcification. C57BL/6 mice were treated for 3 days with high vitamin D (500,000 IU/kg/day) in the presence or absence of recombinant human BMP-7 (rhBMP-7). Expression levels of osteopontin and osteocalcin, markers of the osteoblastic phenotype, were assessed by immunohistochemical staining or Western blotting analysis. Vitamin D increased calcium staining in thoracic aortas and hearts and the expression levels of osteopontin and osteocalcin in mice. Importantly, pretreatment for 7 days and subsequent treatment for 3 days with rhBMP-7 (10 microg/kg/day) abolished the vitamin D-mediated increases in the above parameters. In addition, human aortic smooth muscle cells (HASMCs) were cultured with high beta-glycerophosphate, a phosphate donor, for 2 weeks in the presence or absence of rhBMP-7. High beta-glycerophosphate increased expression levels of osteopontin and osteocalcin as well as calcium staining in HASMCs, but these changes were attenuated by treatment with BMP-7. Thus, BMP-7 inhibits vascular calcification associated with high levels of vitamin D or phosphate. We propose that BMP-7 treatment may be helpful in reducing the risks of cardiovascular disease related to vascular calcification.

Osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL)/receptor activator of NF-κB (RANK) axis, and TNF-related apoptosis-inducing ligand (TRAIL) participate in vascular calcification process including atherosclerosis, but their contributions under high glucose (HG) and phosphate (HP) condition for a long-term period (more than 2 weeks) have not been fully determined. In this study, we evaluated the effects of HG and HP levels over 2 or 4 weeks on the progression of vascular calcification in rat vascular smooth muscle cells (VSMCs). Calcium deposition in VSMCs was increased in medium containing HG (30 mmol/L D-glucose) with β-glycerophosphate (β-GP, 12 mmol/L) after 2 weeks and increased further after 4 weeks. OPG mRNA and protein expressions were unchanged in HG group with or without β-GP after 2 weeks. However, after 4 weeks, OPG mRNA and protein expressions were significantly lower in HG group with β-GP. No significant expression changes were observed in RANKL, RANK, or TRAIL during the experiment. After 4 weeks of treatment in HG group containing β-GP and rhBMP-7, an inhibitor of vascular calcification, OPG expressions were maintained. Furthermore, mRNA expression of alkaline phosphatase (ALP), a marker of vascular mineralization, was lower in the presence of rhBMP-7. These results suggest that low OPG levels after long term HG and phosphate stimulation might reduce the binding of OPG to RANKL and TRAIL, and these changes could increase osteo-inductive VSMC differentiation, especially vascular mineralization reflected by increased ALP activity during vascular calcification.

Runx transcription factors are homologous to the Drosophila paired rule genes runt which has several functions including sex determination, eye development and neurogenesis. In mammals, Runx proteins appear to have genetically rate-limiting functions in a very limited spectrum of tissues and cell types. For example, Runx2 has an essential role for osteoblast differentiation and bone formation. This osteogenesis function does not preclude functions in non-skeletal tissues and cell types, including those of the male reproductive system. In our previous study, hgalactosidase activity was observed in seminiferous tubules of a transgenic LacZ construct driven by 3.0 kb of the Runx2 distal (P1) promoter. In this study, we further explored the possibility of Runx2 expression in murine testis using these Runx2-P1/ LacZ transgenic mice by histology, RT-PCR, and western blot analysis. In histological analysis, the h-galactosidase expression appeared in testis especially seminiferous tubules and epididymis. In a single cell level, h-galactosidase was detected in sperms and not in sertoli or Leydig cells. Indeed, Runx2 expression was observed in isolated mature sperms, which was confirmed by RT-PCR and western blot analysis. Collectively, these results indicate that Runx2 is expressed in sperm of testis. We propose that Runx2 may play an important role during spermatogenesis in mammals.

Interferon-γ (IFN-γ) is well known as a potent inducer in monokine induced by IFN-γ (Mig) mRNA expression. Although lipopolysaccharide (LPS) alone is weakly effective on Mig mRNA expression. the stimulation of LPS and IFN-γ (LPS/IFN-γ simultaneously has been shown to synergize to produce a high level of Mig mRNA in mouse peritoneal macrophages. In this study, interleukin-10 (IL-10) was found to suppress the LPS/IFN-γ-induced Mig mRNA expression in cell type- and mouse strain-specific fashion, but IFN-γ alone-induced Mig mRNA was unaffected by IL-10 under identical experimental conditions. The IL-10-mediated suppression of LPS/IFN-γ-stimulated Mig mRNA expression was dependent on the concentration of IL-10, and was prevented when the agent was added 2 hours after LPS/IFN-γ treatment. The suppressive action of IL-10 was dependent on a protein synthesis. However, IL-10 did not reduce the stability of LPS/IFN-γ-induced Mig mRNA. These data may have important implications for a previously unrecognized role for IL-10 as a regulator of synergistic effect of LPS on the IFN-γ-induced expression of the Mig gene in macrophages. (Immune Network 2002;2(1):12-18)