Miri Bidder

Diet-induced diabetes activates an osteogenic gene regulatory program in the aortas of low density lipoprotein receptor-deficient mice

Vascular calcification is common in people with diabetes and its presence predicts premature mortality. To clarify the underlying mechanisms, we used low density lipoprotein receptor-deficient (LDLR −/−) mice to study vascular calcification in the ascending aorta. LDLR −/− mice on a chow diet did not develop obesity, diabetes, atheroma, or vascular calcification. In contrast, LDLR −/− mice on high fat diets containing cholesterol developed obesity, severe hyperlipidemia, hyperinsulinemic diabetes, and aortic atheroma. A high fat diet without cholesterol also induced obesity and diabetes, but caused only moderate hyperlipidemia and did not result in significant aortic atheroma formation. Regardless of cholesterol content, high fat diets induced mineralization of the proximal aorta (assessed by von Kossa staining) and promoted aortic expression ofMsx2 and Msx1, genes encoding homeodomain transcription factors that regulate mineralization and osseous differentiation programs in the developing skull. Osteopontin(Opn), an osteoblast matrix protein gene also expressed by activated macrophages, was up-regulated in the aorta by these high fat diets. In situ hybridization showed that peri-aortic adventitial cells in high fat-fed mice expressMsx2. Opn was also detected in this adventitial cell population, but in addition was expressed by aortic vascular smooth muscle cells and macrophages of the intimal atheroma. High fat diets associated with hyperinsulinemic diabetes activate an aortic osteoblast transcriptional regulatory program that is independent of intimal atheroma formation. The spatial pattern ofMsx2 and Opn gene expression strongly suggests that vascular calcification, thought to be limited to the media, is an active process that can originate from an osteoprogenitor cell population in the adventitia.

Reciprocal Temporospatial Patterns of Msx2 and Osteocalcin Gene Expression During Murine Odontogenesis

Msx2 is a homeodomain transcription factor that regulates craniofacial development in vivo and osteocalcin (Osc) promoter activity in vitro. Msx2 is expressed in many craniofacial structures prior to embryonic day (E) E14 but is expressed at later stages in a restricted pattern, primarily in developing teeth and the calvarium. We examine Osc expression by in situ hybridization during murine development, detailing temporospatial relationships with Msx2 expression during preappositional and appositional odontogenesis and calvarial osteogenesis. Osc expression at E14–14.5 is very low, limited to a few perichondrial osteoblasts in the dorsal aspect of developing ribs. At E16.5 and E18.5, Osc expression is much higher, widely expressed in skeletal osteoblasts, including calvarial osteoblasts that do not express Msx2. No Osc is detected in early preappositional teeth that express Msx2. In incisors studied at an early appositional phase, Msx2 is widely expressed in the tooth, primarily in ovoid preodontoblasts and subjacent dental papilla cells. Osc is detected only in a small number of maturing odontoblasts that also express α1(I) collagen (Col1a1) and that are postproliferative (do not express histone H4). Msx2 expression greatly overlaps both histone H4 and Col1a1 expression in ovoid preodontoblasts and dental papilla cells. By the late appositional phases of E18.5 and neonatal teeth, Osc mRNA is highly expressed in mature columnar odontoblasts adjacent to accumulating dentin. In appositional bell‐stage molars, reciprocal patterns of Msx2 and Osc are observed in adjacent preodontoblasts and odontoblasts within the same tooth. Osc is expressed in mature columnar odontoblasts, while Msx2 is expressed in adjacent immature ovoid preodontoblasts. In less mature teeth populated only by immature ovoid preodontoblasts, only Msx2 is expressed‐–no Osc is detected. Thus, Msx2 and Osc are expressed in reciprocal patterns during craniofacial development in vivo, and Msx2 expression in preodontoblasts clearly preceeds Osc expression in odontoblasts. In functional studies using MC3T3‐E1 calvarial osteoblasts, Msx2 suppresses endogeneous Osc, but not osteopontin, mRNA accumulation. In toto, these data suggest that Msx2 suppresses Osc expression in the craniofacial skeleton at stages immediately preceeding odontoblast and osteoblast terminal differentiation.

Aberrant promoter methylation and silencing of the POU2F3 gene in cervical cancer

POU2F3 (OCT11, Skn-1a) is a keratinocyte-specific POU transcription factor whose expression is tied to squamous epithelial stratification. It is also a candidate tumor suppressor gene in cervical cancer (CC) because it lies in a critical loss of heterozygosity region on11q23.3 in that cancer, and its expression is lost in more than 50% of CC tumors and cell lines. We now report that the loss of POU2F3 expression is tied to the hypermethylation of CpG islands in the POU2F3 promoter. Bisulfite sequencing analysis revealed that methylation of specific CpG sites (−287 to −70 bp) correlated with POU2F3 expression, which could be reactivated with a demethylating agent. Combined bisulfite restriction analysis revealed aberrant methylation of the POU2F3 promoter in 18 of 46 (39%) cervical tumors but never in normal epithelium. POU2F3 expression was downregulated and inversely correlated with promoter hypermethylation in 10 out of 11 CC cell lines. Immunohistochemical analysis on a cervical tissue microarray detected POU2F3 protein in the epithelium above the basal layer. As the disease progressed, expression also decreased, especially in invasive squamous cell cancer (70% loss). Thus, aberrant DNA methylation of the CpG island in POU2F3 promoter appears to play a key role in silencing this gene expression in human CC. The results suggested that POU2F3 might be one of the CC-related tumor suppressor genes, which are disrupted by both epigenetic and genetic mechanisms.

Ets Domain Transcription Factor PE1 Suppresses Human Interstitial Collagenase Promoter Activity by Antagonizing Protein−DNA Interactions at a Critical AP1 Element†

In MC3T3E1 calvarial osteoblasts, fibroblast growth factor receptor (FGFR) signaling elicits multiple transcriptional responses, including upregulation of the interstitial collagenase/matrix metalloproteinase 1 (MMP1) promoter. FGF responsiveness maps to a bipartite Ets/AP1 element at base pairs -123 to -61 in the human MMP1 promoter. Under basal conditions, the MMP1 promoter is repressed in part via protein-DNA interactions at the Ets cognate, and minimally two mechanisms convey MMP1 promoter upregulation by FGF2: (a) transcriptional activation via Fra1/c-Jun containing DNA-protein interactions at the AP1 cognate and (b) derepression of promoter activity regulated by the Ets cognate. To identify osteoblast Ets repressors that potentially participate in gene expression in the osteoblast, we performed reverse transcription-polymerase chain reaction (RT-PCR) analysis of mRNA isolated from MC3T3E1 cells, using degenerative amplimers to the conserved Ets DNA binding domain to survey the Ets genes expressed by these cells. Six distinct Ets mRNAs were identified: Ets2, Fli1, GABPalpha, SAP1, Elk1, and PE1. Of these, only PE1 has extensive homology to the known Ras-regulated Ets transcriptional repressor, ERF. Therefore, we cloned and characterized PE1 cDNA from a mouse brain library and performed functional analysis of this particular Ets family member. A 2 kb transcript was isolated from brain that encodes a approximately 57 kDa protein; the predicted protein contains the known N-terminal Ets domain of PE1 and a novel C-terminal domain with signficant homology to murine ERF. The murine PE1 open reading frame (ORF) is much larger than the previously reported human PE1 ORF. Consistent with this, affinity-purified rabbit anti-mouse PE1 antibody specifically recognizes an approximately 66 kDa protein present only in the nuclear fraction of MC3T3E1 osteoblasts. Recombinant PE1 binds authentic AGGAWG Ets DNA cognates, and transient transfection studies demonstrate that PE1 represses MMP1 promoter activity. Surprisingly, although deletion of the MMP1 Ets cognate at nucleotides -88 to -83 abrogates FGF2 induction, it does not prevent suppression of the AP1-dependent MMP1 promoter by PE1. PE1 regulation maps to the MMP1 promoter region -75 to -61, suggesting that PE1 suppresses transcription via protein-protein interactions with AP1. Consistent with this, recombinant GST-PE1 specifically inhibits the formation of protein-DNA interactions on the MMP1 AP1 site (-72 to -66) when present in an admixture with MC3T3E1 crude nuclear extract. In toto, these data indicate that PE1 participates in the transcriptional regulation of the MMP1 promoter in osteoblasts. As observed with other transcriptional repressors of MMP1 gene expression, transcriptional suppression by PE1 occurs via inhibition of AP1-dependent promoter activity.

Expression of mRNA for Vascular Endothelial Growth Factor at the Repair Site of Healing Canine Flexor Tendon

Neovascularization is an important and prominent feature of tendon healing that contributes to wound repair and potentially to adhesion formation. To define the location of cell populations that recruit and organize the angiogenic response during early healing of flexor tendon, we examined the gene expression pattern of the prototypic angiogenic factor, vascular endothelial growth factor, at and around the tenorrhaphy site in a canine model of flexor tendon repair. In situ hybridization with radiolabeled antisense riboprobes was used to identify tendon cell populations that contribute to the neovascularization process by expressing vascular endothelial growth factor and to relate this cell population to the previously described cell populations that participate in matrix synthesis (express type alpha1(I) collagen) and mitotic renewal (express histone H4). The majority of cells (approximately 67%) within the repair site itself express vascular endothelial growth factor mRNA; however, minimal levels accumulate within cells of the epitenon (approximately 10% of cells; p < 0.0002). By contrast, expression of type alpha1(I) collagen and histone H4 does not differ significantly between the epitenon and the repair site (uniformly approximately 30% of cells). Thus, a gradient of cell populations expressing vascular endothelial growth factor exists in the repairing tendon. These data suggest a potential contribution of cells within the repair site to the organization of angiogenesis during the early postoperative phase of tendon healing.