ZEB1 regulates bone metabolism in osteoporotic rats through inducing POLDIP2 transcription.

BACKGROUND: Osteoporosis (OP) is a common metabolic bone disease mainly involving bone remodeling and blood vessels. The current study aimed to explore the role of zinc finger E-box binding homeobox 1 (ZEB1) in OP. METHODS: First, gene expression microarrays for OP were downloaded from the Gene Expression Omnibus database and analyzed to screen for potential targets. Subsequently, a rat OP model was constructed using ovariectomy (OVX), and osteoblastic and osteoclastic differentiation and alterations in osteoporotic symptoms were observed upon intraperitoneal injection of oe-ZEB1 lentiviral vectors. DNA polymerase delta interacting protein 2 (POLDIP2) was predicted to be a downstream target of ZEB1, which was validated by ChIP-qPCR and dual-luciferase experiments. RAW264.7 cells were subjected to lentiviral vector infection of oe-ZEB1 and/or sh-POLDIP2, followed by RANKL treatment to induce osteoclast differentiation. RESULTS: ZEB1 was poorly expressed in blood samples of postmenopausal patients with OP and in bone tissues of OVX-treated rats. Overexpression of ZEB1 or POLDIP2 in OVX rats promoted osteoblastogenesis and inhibited osteoclast differentiation. In RANKL-treated RAW264.7 cells, the transcription factor ZEB1 enhanced the expression of POLDIP2, and silencing of POLDIP2 attenuated the inhibitory effect of oe-ZEB1 on the differentiation of macrophages RAW264.7 to osteoclasts. CONCLUSIONS: ZEB1 promotes osteoblastogenesis and represses osteoclast differentiation, ultimately reducing the occurrence of postmenopausal OP by elevating the expression of POLDIP2.

Cloning of two rat PDIP1 related genes and their interactions with proliferating cell nuclear antigen.

Human polymerase delta-interacting protein 1 (PDIP1) is a tumor necrosis factor alpha and interleukin 6 inducible protein that interacts directly with proliferating cell nuclear antigen (PCNA) and the small subunit (p50) of DNA polymerase delta. PDIP1 binds PCNA and p50 simultaneously and stimulates polymerase delta activity in vitro in the presence, but not the absence, of PCNA. It has been suggested that PDIP1 provides a link between cytokine activation and DNA replication in eukaryotes. Here these authors report the cloning of two rat genes homologous to human PDIP1, termed rat PDIP1 and rat tumor necrosis factor-induced protein 1 (TNFAIP1). The rat PDIP1 is mapped to chromosome 1q36 cM region, spans approximately 18.7 kb, and is organized into six exons. The rat TNFAIP1 gene is mapped to chromosome 10q25 cM, spans approximately 12.9 kb, and is composed of seven exons. The deduced proteins of rat PDIP1 and rat TNFAIP1 share 63.1% sequence identity with each other and are highly conserved in the majority of the middle portion of the two proteins, which encode a BTB/POZ domain at the N-terminus and a PCNA binding motif (QTKV-EFP) at the C-terminus, respectively. The BTB / POZ domain and the PCNA binding motif are highly conserved during the evolution. Both rat PDIP1 and rat TNFAIP1 were demonstrated to interact with PCNA via BIAcore, GST pull-down, and co-immunoprecipitation assays. Like the human PDIP1, both rat PDIP1 and rat TNFAIP1 stimulate polymerase delta activity in vitro in a PCNA-dependent way.

Comparison of the efficiency of synthesis past single bulky DNA adducts in vivo and in vitro by the polymerase III holoenzyme.

Previous studies from our laboratory revealed that site-specific and stereospecific styrene oxide (SO) lesions in M13 DNA were readily bypassed when transfected into Escherichia coli cells, but these same lesions blocked the progress of several purified polymerases in vitro when situated in oligodeoxynucleotide templates (Latham, G. J., et al. (1993) J. Biol. Chem. 268, 23427-23434; Latham, G. J., et al. (1995) Chem. Res. Toxicol. 8, 422-430). To resolve this apparent discrepancy, we constructed single-stranded M13 genomes containing single SO adducts and compared their replication efficiencies in E. coli cells to the extent of bypass synthesis in vitro using three different complexes of the purified E. coli polymerase III (Pol III) holoenzyme. The transformation efficiencies of the SO-adducted M13 templates were comparable to those of the nonadducted controls, indicating facile bypass in E. coli. When the identical adducted M13 vectors were replicated in vitro with the reconstituted complexes of the Pol III holoenzyme, the results were consistent with the in vivo data: Synthesis past two of the three SO adducts in M13 was unhindered relative to synthesis on the unadducted M13 control template. Since our previous in vitro assays indicated that SO adducts in 33-mer templates largely blocked polymerases other than Pol III, we repeated these studies using reconstituted Pol III. Significantly, Pol III replication was poorly processive and strongly terminated by SO lesions in 33-mer templates. This result was in stark contrast to the efficient bypass in vitro of the same adducts in M13 DNA. In fact, Pol III-mediated bypass was enhanced to > 75-fold on adducted circular M13 templates as compared to adducted linear oligodeoxynucleotides. The implications of the effects of polymerase processivity and template-primer structure upon lesion bypass are discussed.

Interaction of a folded chromosome-associated protein with single-stranded DNA-binding protein of Escherichia coli, identified by affinity chromatography.

A single-stranded DNA-binding protein (SSB) affinity column was prepared by optimizing the coupling of Escherichia coli single-stranded DNA-binding protein to Affi-Gel 10. The bound SSB retained its ability to specifically bind single-stranded DNA. When nuclease-treated cell extracts were incubated with the SSB beads overnight at 4 degrees C, a major protein of Mr = 25,000 was bound. At shorter incubation times, two additional proteins of Mr = 32,000 and 36,000 were also detected. In the absence of nuclease treatment, eight additional proteins ranging from Mr = 14,000 to 160,000 also bound to the affinity column. The major Mr = 25,000 protein has been shown to be a folded chromosome-associated protein. Its binding to SSB is strongly enhanced by the addition of DNA polymerase III or DNA polymerase III holoenzyme.