Faculty Bibliography

Histone deacetylases (HDACs) are crucial regulators of gene expression in transcriptional co-repressor complexes. Previously, we reported that HDAC4 was a basal repressor of matrix metalloproteinase-13 (MMP-13) transcription and parathyroid hormone (PTH) regulates HDAC4 to control MMP-13 promoter activity through causing its dissociation from Runx2. The aim of the present study was to determine the mechanism and signal transduction pathway by which this occurred. We found that PTH induces the protein kinase A (PKA)-dependent phosphorylation of HDAC4 in the nucleus of cells of the rat osteoblastic cell line, UMR 106-01. In contrast, PKA is necessary for the translocation of HDAC4 into the nucleus. We demonstrated that PKA-dependent phosphorylated HDAC4 is released from Runx2 on the runt domain of the MMP-13 promoter in these cells. Point mutations of serines at 355, 576, 740 or threonine at 815 in rat HDAC4 showed that only mutation at serine 740 prevented the release of HDAC4 from Runx2 on the MMP-13 promoter and also the PTH stimulation of MMP-13 transcription. Using antibodies to the equivalent phosphorylation site in human HDAC4 (serine 632), we found that PTH transiently stimulated phosphorylation of this residue and this was abolished when PKA was inhibited. Thus, PTH-induced PKA phosphorylation of serine 740 in rat HDAC4 is crucial for regulating MMP-13 transcription in osteoblasts. The protein 14-3-3 beta is associated with the shuttling of HDAC4 between nucleus and cytoplasm. Colocalization of 14-3-3 beta and HDAC4 is increased after PTH stimulation and both accumulate in the cytoplasm. These mechanisms regulating HDAC4 and their roles in such processes are crucial for bone and chondrocyte development. Our data support a link between PTH regulating HDAC4 phosphorylation by PKA and trafficking and the control of MMP-13 transcription through association with Runx2

The epidermal growth factor receptor (EGFR) and its ligands regulate key processes of cell biology, such as proliferation, survival, differentiation, migration, and tumorigenesis. We previously showed that, EGFR signaling pathway is an important bone regulator and it primarily plays an anabolic role in bone metabolism. In this study, we demonstrated that EGF-like ligands strongly inhibited osteoblast differentiation and mineralization in several lines of osteoblastic cells. Real-time RT-PCR and promoter reporter assays revealed that EGF-like ligands suppressed the expression of both early and late bone marker genes at the transcriptional level in the differentiating osteoblasts via an EGFR-dependent manner. This inhibitory effect of EGFR signaling was not dependent on its mitogenic activity. Furthermore, we demonstrated that EGFR signaling reduced the expression of two major osteoblastic transcription factors Runx2 (type II) and Osterix in osteoblast differentiating cells. EGFR-induced decrease in Runx2 transcriptional activity was confirmed by Runx2 reporter and chromatin immunoprecipitation assays. EGFR signaling increased the protein amounts of transcription co-repressors HDAC4 and 6 and over-expression of HDAC4 decreased Runx2 amount in differentiating osteoblasts, implying that HDACs contribute to the down-regulation of Runx2 by EGFR. Moreover, activation of EGFR in undifferentiated osteoprogenitors attenuated the expression of early bone markers and Osterix and decreased Runx2 protein amounts. Together with our previous data, that EGFR stimulates osteoprogenitor proliferation and that blocking EGFR activity in osteoblast lineage cells results in fewer osteoprogenitors and an osteopenic phenotype, we conclude that EGFR signaling is important for maintaining osteoprogenitor population at an undifferentiated stage.

The current medical examinations for detecting endometrial cancer can sometimes be stressful and inconvenient for examinees and examiners. Therefore, we attempted to develop an autoscan-virtual cytology system for detecting endometrial cancer without relying on judgment by the human eye. Exfoliated cells from the uterus were retrieved using a tampon inserted for 3 h. More than 100 monoclonal antibodies (mAb) developed by us were screened in three steps of immunohistochemistry to find mAb sets that would enable the cancer and normal endometrium to be perfectly distinguished. The exfoliated cells provided by 30 endometrial cancer patients and a total of 37 samples of 14 non-malignant volunteers including the menstrual cycle were analyzed using imaging cytometry. All samples contained epithelial cells and dysplasia cells, but the pathologist could not definitively diagnose all of them as endometrial cancer cells because most cells had degenerated. Twenty-two of 28 endometrial cancer tissues (79%) were positive with four mAb sets, CRELD1, GRK5, SLC25A27 and STC2, and 22 of 22 normal endometriums (100%) were negative. Our newly developed autoscan-virtual cytology for exfoliated endometrial cells showed overall sensitivity for endometrial cancer patients and overall specificity for volunteers of 50% (15/30) and 95% (35/37), respectively. Our autoscan-virtual cytology combined with cancer-specific mAb and imaging cytometry could be useful for endometrial cancer detection. Autoscan-virtual cytology for endometrial cancer deserves further evaluation for future endometrial cancer screening.

Parathyroid hormone (PTH) is a hormone regulating bone remodeling through its actions on both bone formation and bone resorption. Previously we reported that PTH induces matrix metalloproteinase-13 (MMP-13) transcription in osteoblastic cells. Here, we show that histone deacetylase 4 (HDAC4) interacts with Runx2, binds the MMP-13 promoter, and suppresses MMP-13 gene transcription in the rat osteoblastic cell line, UMR 106-01. PTH induces the rapid cAMP-dependent protein kinase-dependent release of HDAC4 from the MMP-13 promoter and subsequent transcription of MMP-13. Knock-out of HDAC4 either by siRNA in vitro or by gene deletion in vivo leads to an increase in MMP-13 expression, and overexpression of HDAC4 decreases the PTH induction of MMP-13. All of these observations indicate that HDAC4 represses MMP-13 gene transcription in bone. Moreover, PTH stimulates HDAC4 gene expression and enzymatic activity at times corresponding to the reassociation of HDAC4 with the MMP-13 promoter and a decline in its transcription. Thus, HDAC4 is a basal repressor of MMP-13 transcription, and PTH regulates HDAC4 to control MMP-13 promoter activity. These data identify a novel and discrete mechanism of regulating HDAC4 levels and, subsequently, gene expression.

Parathyroid hormone (PTH) is the major hormone regulating calcium homeostasis, but it also has a significant role as an anabolic hormone in bone when administered by intermittent injection. The mechanisms for the bone anabolic effect of PTH are not understood. However, it has been established that the anabolic effects of PTH require osteoclast activity. PTH acts through a single receptor on the osteoblast to regulate many known genes. Previous microarray studies in our laboratory have shown that the most highly regulated gene, monocyte chemoattractant protein-1 (MCP-1), is rapidly and transiently induced when PTH(1-34) is injected intermittently in rats for 14 days. Through further in vivo studies, we found that rats treated with PTH(1-34) for 14 days showed a significant increase in serum MCP-1 levels 2 h after PTH injection (159 pg/ml) compared to basal levels obtained prior to the last injection (65 pg/ml). Furthermore, serum MCP-1 levels increase with the duration of treatment. Compared to vehicle treatment, PTH(1-34) treatment in rats for 1, 7 and 14 days showed a 24%, 53% and 79% increase in serum MCP-1 levels 2 h after injection, respectively. Using immunohistochemistry, increased MCP-1 expression is evident in trabecular osteoblasts by the second PTH(1-34) injection with peak staining occurring between 3 and 7 days of treatment. Male and female MCP-1 knockout mice injected daily with 80 ug/kg PTH(1-34) for 6 weeks showed significantly reduced anabolic effects of PTH compared to wild type mice as measured by pQCT and micro-CT. PTH-treated male MCP-1 null mice have significantly less trabecular bone mineral density (means, mg/cm³, vehicle: 454; PTH: 486) than the corresponding PTH-treated wild type mice (means, mg/cm³, vehicle: 452; PTH: 521). Trabecular bone volume (BV/TV) increased 27% in PTH-treated wild type mice with a corresponding increase in trabecular thickness (Tb.Th), however no increase in BV/TV or Tb.Th was observed in the PTH-treated MCP-1 null mice. Hi!

PTH regulates transcription of a number of genes involved in bone remodeling and calcium homeostasis. We have previously shown that the matrix metalloproteinase-13 (MMP-13) gene is induced by PTH in osteoblastic cells as a secondary response through the protein kinase A pathway requiring the runt domain and activator protein 1 binding sites of the proximal promoter. Here, we investigated the changes PTH causes in histone acetylation in this region (which contains the only deoxyribonuclease-hypersensitive sites in the promoter) leading to MMP-13 gene activation in these cells. Chromatin immunoprecipitation experiments revealed that PTH rapidly increased histone H4 acetylation followed by histone H3 acetylation associated with the different regions of the MMP-13 proximal promoter. The hormone also stimulated p300 histone acetyl transferase activity and increased p300 bound to the MMP-13 proximal promoter, and this required protein synthesis. Upon PTH treatment, Runx2, already bound to the runt domain site of the MMP-13 promoter, interacted with p300, which then acetylated histones H4 and H3. The knockdown of either Runx2 or p300 by RNA interference reduced PTH-induced acetylation of histones H3 and H4, association of p300 with the MMP-13 promoter, and resultant MMP-13 gene transcription. Overall, our studies suggest that without altering the gross chromatin structure, PTH stimulates acetylation of histones H3 and H4 via recruitment of p300 to Runx2 bound to the MMP-13 promoter, resulting in gene activation. This work establishes the molecular basis of transcriptional regulation in osteoblasts by PTH, a hormone acting through a G-protein coupled receptor

Matrix metalloproteinase-13 (MMP-13) plays a critical role in parathyroid hormone (PTH)-induced bone resorption. PTH acts via protein kinase A (PKA) to phosphorylate and stimulate the transactivation of Runx2 for MMP-13 promoter activation. We show here that PTH stimulated Runx2 phosphorylation in rat osteoblastic cells. Runx2 was phosphorylated on serine 28 and threonine 340 after 8-bromo cyclic adenosine mono phosphate (8-Br-cAMP) treatment. We further demonstrate that in the presence of 8-Br-cAMP, the wild-type Runx2 construct stimulated MMP-13 promoter activity, while the Runx2 construct having mutations at three phosphorylation sites (S28, S347 and T340) was unable to stimulate MMP-13 promoter activity. Thus, we have identified the Runx2 phosphorylation sites necessary for PKA stimulated MMP-13 promoter activation and this event may be critical for bone remodeling

Interleukin-18 (IL-18) can regulate osteoblast and osteoclast function. We have identified, using cDNA microarray technology, that IL-18 expression is increased in UMR 106-01 rat osteoblastic cells in response to parathyroid hormone (PTH) treatment. Confirmation of these data using real-time reverse transcription-PCR showed that steady-state levels of IL-18 mRNA increased by 2 h (3-fold), peaked by 4 h (10-fold), and had diminished after 12 h (4.4-fold) and that this regulation was via the protein kinase A signaling pathway and did not involve activation of the PKC signal cascade. PTH regulation of IL-18 was confirmed at the protein level, and analysis of differentiating primary rat calvarial osteoblasts verified that both IL-18 mRNA and protein are regulated by PTH in primary rat osteoblasts. Promoter reporter assays revealed that PTH regulated the upstream IL-18 promoter and induced the exon 1 containing 1.1-kb IL-18 mRNA transcript in primary osteoblast cells. The in vivo physiological role of IL-18 in the anabolic actions of PTH on bone was then assessed using IL-18 knock-out mice. Female IL-18 null mice and wild-type littermate controls were injected with vehicle or 8 microg/100 g of human 1-38 PTH for 4 weeks. In IL-18 knock-out animals the anabolic effect of PTH (determined by bone mineral density changes in the proximal tibia) was abolished in trabecular bone but not in the cortical component. These data characterize the PTH regulation of IL-18 expression in osteoblastic cells and suggest that this cytokine is involved in the anabolic actions of PTH

Parathyroid hormone (PTH) functions as an essential regulator of calcium homeostasis and as a mediator of bone remodeling. We have already shown that PTH stimulates the expression of matrix metalloproteinase-13 (MMP-13), which is responsible for degrading components of extracellular matrix. We have hypothesized that histone deacetylases (HDACs) are involved with PTH-induced MMP-13 gene expression in the osteoblastic cell line, UMR 106-01. We have shown that PTH profoundly regulates HDAC4 in UMR 106-01 cells through a PKA-dependent pathway, leading to removal of HDAC4 from the MMP-13 promoter and its enhanced transcription. Understanding the mechanism of how HDACs affect osteoblast differentiation and mineralization will identify new theraupeutic methods for bone diseases, such as osteoporosis and multiple myeloma