Lan Pham

University of Minnesota Duluth, Duluth, Minnesota, United States

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Publications (14)31.79 Total impact

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    ABSTRACT: To investigate the role of BMP signaling in osteoclastogenesis in vivo, we eliminated BMPRII in osteoclasts by creating a BMPRIIfl/fl;lysM-Cre mouse strain. cKO mice are osteopetrotic compared to WT controls, due to a decrease in osteoclast activity. Bone marrow macrophages (BMMs) isolated from cKO mice are severely inhibited in their capacity to differentiate into mature osteoclasts in the presence of M CSF and RANK ligand. We also show that BMP non canonical (MAPK) and canonical (SMAD) pathways are utilized at different stages of osteoclast differentiation. BMP2 induces p38 phosphorylation in pre fusion osteoclasts and increases SMAD phosphorylation around osteoclast precursor fusion. Phosphorylation of MAPKs, but not SMADs, was decreased in differentiated BMMs from cKO animals. Treating BMMs with the SMAD inhibitor dorsomorphin confirms the requirement for the canonical pathway around the time of fusion. These results demonstrate the requirement for BMP signaling in osteoclasts for proper bone homeostasis, and also explore the complex signaling mechanisms employed by BMP signaling during osteoclast differentiation.
    Journal of Biological Chemistry 11/2013; · 4.65 Impact Factor
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    ABSTRACT: Objectives: Bone Morphogenetic Proteins (BMPs) are key regulators of bone development and are increasingly used in clinical settings to improve fracture healing and fixation of implanted structures. Despite this emerging use, understanding of the regulation of BMP functions remains limited. Twisted gastrulation (TWSG1) is an extracellular BMP binding protein, which serves to modulate BMP signaling. Mice carrying deletion of exon 4 of TWSG1 have a range of craniofacial defects, including micrognathia and agnathia as well as osteopenia. We hypothesized that glycosylation of TWSG1 plays a key role in the interaction with BMPs and regulating their activity. Methods: Glycosylation sites in TWSG1 were predicted using the EnsemblGly software and confirmed by site-directed mutagenesis and enzymatic deglycosylation, followed by western blotting. Interaction of BMPs with TWSG1 was assayed by immunoprecipitation with wild type TWSG1 and TWSG1 that had mutated or absent glycosylation sites. Non-glycosylated and glycosylated recombinant TWSG1 proteins were generated in bacterial and insect cell expression systems, respectively, and assayed quantitatively for BMP binding using surface plasmon resonance analysis. A mandibular explant culture system was used to examine the effect of these TWSG1 proteins on the expression of the BMP target gene Msx2. Results: TWSG1 in mice has two glycosylation sites, which are both encoded by the fourth exon. Deletion of the entire exon 4 or mutation of both glycosylation sites abolishes glycosylation of mTWSG1. Constructs with mutated glycosylation sites have significantly reduced BMP binding activity. A non-glycosylated form of the protein binds to BMPs with approximately 10-fold reduced affinity compared to the glycosylated form. The non-glycosylated form of TWSG1 is unable to suppress Msx2 expression in mandibular explants, while glycosylated forms do suppress Msx2. Conclusions: We report that glycosylation is essential for normal action of TWSG1 and hence may represent an important variable in the regulation of BMP signaling.
    IADR General Session 2012; 06/2012
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    ABSTRACT: Histone deacetylases (HDACs) are negative regulators of transcription. Endochondral bone formation including chondrocyte and osteoblast maturation is regulated by HDACs. Very little is known about the role HDACs play in osteoclast differentiation. It has been previously reported that HDAC inhibitors, trichostatin A and sodium butyrate, suppress osteoclast differentiation through multiple mechanisms. In this study, we report that suppression of HDAC3 expression similar to HDAC inhibitors inhibits osteoclast differentiation, whereas osteoclasts suppressed for HDAC7 expression had accelerated differentiation when compared with control cells. Mitf, a transcription factor, is necessary for osteoclast differentiation. We demonstrate that Mitf and HDAC7 interact in RAW 264 cells and osteoclasts. The transcriptional activity of Mitf is repressed by HDAC7. Lastly, we show that either the amino or the carboxyl terminus of HDAC7 is sufficient for transcriptional repression and that the repression of HDAC7 is insensitive to trichostatin A, indicating that HDAC7 represses Mitf at least in part by deacetylation-independent mechanism.
    Journal of Biological Chemistry 04/2011; 286(14):12056-12065. · 4.65 Impact Factor
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    ABSTRACT: Histone deacetylases (HDACs) are negative regulators of transcription and influence endochondral bone formation. A number of HDACs regulate chondrocyte and osteoblast differentiation and activity however, little is known about the role HDACs play in osteoclast differentiation. Studying how HDACs affect osteoclasts function will increase our understanding of skeletal development, maintenance, and pathological states. Objective: Determine the significance of HDAC regulation on osteoclastogenesis. Methods: Murine osteoclast precursors were isolated from bone marrow and differentiated in-vitro. Viral vectors were used to suppress expression of HDAC3 and HDAC7. Osteoclast differentiation was assessed by TRAP staining. The expression of osteoclast makers were measured by qPCR. Mitf-HDAC7 interactions were assayed by immunoprecipitation and western blot. Results: Suppression of HDAC3 by shRNA closely mirrors the inhibitory effect of non-specific HDAC inhibitors on osteoclast formation while osteoclasts suppressed for HDAC7 expression had accelerated differentiation. Conclusions: We demonstrated that HDAC7 inhibits osteoclast differentiation by interacting with Mitf, a transcription factor necessary for osteoclast differentiation, and treatment with RANKL disrupts the Mitf-HDAC7 interaction, allowing Mitf-target gene expression and osteoclastic differentiation. Lastly, we show that the repression of Mitf by HDAC7 is deacetylation-independent. (NIH/NIDCR: T32DE007288(L.P.))
    IADR General Session 2011; 03/2011
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    ABSTRACT: Bone morphogenetic proteins (BMPs) have been shown to regulate both osteoblasts and osteoclasts. We previously reported that BMP2 could directly enhance RANKL-mediated osteoclast differentiation by increasing the size and number of osteoclasts. Similarly, genetic deletion of the BMP antagonist Twisted gastrulation (TWSG1) in mice, resulted in an enhancement of osteoclast formation, activity and osteopenia. This was accompanied by increased levels of phosphorylated Smad (pSmad) 1/5/8 in Twsg1(-/-) osteoclasts in vitro. The purpose of this study was to develop an adenoviral vector overexpressing Twsg1 as a means of inhibiting osteoclast activity. We demonstrate that overexpressing TWSG1 in primary osteoclasts decreased the size and number of multinuclear TRAP-positive osteoclasts, expression of osteoclast genes, and resorption ability. Overexpression of TWSG1 did not affect osteoclast proliferation or apoptosis. However, overexpression of TWSG1 decreased the levels of pSmad 1/5/8 in osteoclasts. Addition of exogenous BMP2 to osteoclasts overexpressing TWSG1 rescued the size and levels of pSmad 1/5/8 compared to cultures infected with a control virus. Finally, TWSG1 overexpression in osteoclasts isolated from the Twsg1(-/-) mice rescued size of the osteoclasts while further addition of exogenous BMP2 reversed the effect of TWSG1 overexpression and increased the size of the osteoclasts similar to control virus infected cells. Taken together, we demonstrate that overexpressing TWSG1 in osteoclasts via an adenoviral vector results in inhibition of osteoclastogenesis and may provide a potential therapy for inhibiting osteoclast activity in a localized manner.
    Journal of Cellular Biochemistry 03/2011; 112(3):793-803. · 3.06 Impact Factor
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    ABSTRACT: Histone deacetylases (HDACs) are negative regulators of transcription. Endochondral bone formation including chondrocyte and osteoblast maturation is regulated by HDACs. Very little is known about the role HDACs play in osteoclast differentiation. It has been previously reported that HDAC inhibitors, trichostatin A and sodium butyrate, suppress osteoclast differentiation through multiple mechanisms. In this study, we report that suppression of HDAC3 expression similar to HDAC inhibitors inhibits osteoclast differentiation, whereas osteoclasts suppressed for HDAC7 expression had accelerated differentiation when compared with control cells. Mitf, a transcription factor, is necessary for osteoclast differentiation. We demonstrate that Mitf and HDAC7 interact in RAW 264 cells and osteoclasts. The transcriptional activity of Mitf is repressed by HDAC7. Lastly, we show that either the amino or the carboxyl terminus of HDAC7 is sufficient for transcriptional repression and that the repression of HDAC7 is insensitive to trichostatin A, indicating that HDAC7 represses Mitf at least in part by deacetylation-independent mechanism.
    Journal of Biological Chemistry 02/2011; 286(14):12056-65. · 4.65 Impact Factor
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    ABSTRACT: In response to anecdotal reports that African American neighborhoods are targeted for high-alcohol malt liquor advertising, the authors observed alcohol ads on off-premise alcohol outlets, billboards, and transit structures in 10 U.S. cities over 3 years. Malt liquor ads were prevalent on storefronts, but rare on billboards. Using Poisson regression, the authors found that storefront malt liquor ads were more common in neighborhoods with higher percentages of African Americans, even after controlling for social and physical disorder. Results suggest that policymakers attempting to reduce malt liquor-related harms may do well to consider regulations that limit storefront advertising exposure.
    Journal of Ethnicity in Substance Abuse 01/2011; 10(1):24-38.
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    ABSTRACT: Twisted gastrulation (TWSG1) is a conserved, secreted glycoprotein that modulates signaling of bone morphogenetic proteins (BMPs) in the extracellular space. Deletion of exon 4 of mouse Twsg1 (mTwsg1) is associated with significant craniofacial defects. However, little is understood about the biochemical properties of the corresponding region of the protein. We have uncovered a significant role for exon 4 sequences as encoding the only two glycosylation sites of the mTWSG1 protein. Deletion of the entire exon 4 or mutation of both glycosylation sites within exon 4 abolishes glycosylation of mTWSG1. Importantly, we find that constructs with mutated glycosylation sites have significantly reduced BMP binding activity. We further show that glycosylation and activity of TWSG1 recombinant proteins vary markedly by cellular source. Non-glycosylated mTWSG1 made in E. coli has both reduced affinity for BMPs, as shown by surface plasmon resonance analysis, and reduced BMP inhibitory activity in a mandibular explant culture system compared to glycosylated proteins made in insect cells or murine myeloma cells. This study highlights an essential role for glycosylation in Twisted gastrulation action.
    Frontiers in Physiology 01/2011; 2:59.
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    ABSTRACT: Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF-kappaB ligand (RANKL) to enhance in vitro differentiation of osteoclast-like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow-derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p-SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p-SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL-stimulated formation of multinuclear TRAP-positive cells. The BMP antagonist noggin inhibited RANKL-mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts-like cells.
    Journal of Cellular Biochemistry 03/2010; 109(4):672-82. · 3.06 Impact Factor
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    ABSTRACT: There is strong clinical evidence that implicates tenofovir in the loss of bone mineral density during treatment of human immunodeficiency virus infection. In this study, we sought to test the hypothesis that tenofovir treatment of osteoblasts causes changes in the gene expression profile that would impact osteoblast function during bone formation. Primary osteoblasts were isolated and then treated with the tenofovir prodrug, tenofovir disoproxil fumarate (TDF). Total RNA from TDF-treated and untreated osteoblasts were extracted and used for microarray analysis to assess TDF-associated changes in the gene expression profile. Strikingly, the changes in gene expression profiles involved in cell signaling, cell cycle and amino acid metabolism, which would likely impact osteoblast function in bone formation. Our findings demonstrate for the first time that tenofovir treatment of primary osteoblasts results in gene expression changes that implicate loss of osteoblast function in tenofovir-associated bone mineral density loss.
    Biochemical and Biophysical Research Communications 02/2010; 394(1):48-53. · 2.28 Impact Factor
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    ABSTRACT: Clinical observations have revealed a strong correlation between loss of bone density in HIV-infected individuals, particularly in conjunction with the antiretroviral drug tenofovir, a nucleotide analog that inhibits HIV reverse transcriptase. The most compelling correlations have been observed in clinical studies involving young children and adolescents. These observations strongly suggest that bone density is being affected during active bone growth and development, implicating a role for tenofovir in bone loss. Here we discuss the literature and potential mechanisms for how tenofovir-associated bone loss may arise, which likely involves perturbation of cellular DNA synthesis and gene expression. Elucidation of the mechanism(s) involved in tenofovir-mediated bone loss will help in developing adjuvant therapies to reduce tenofovir-associated bone density loss.
    Therapeutics and Clinical Risk Management 01/2010; 6:41-7.
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    ABSTRACT: Clinical observations have implicated the antiretroviral drug tenofovir with bone density loss during the management of HIV infection. The goal of this study was to investigate the in vitro effects of tenofovir exposure of primary osteoclasts in order to gain insights into the potential mechanisms for the drug-induced bone density loss. We hypothesized that tenofovir may alter the expression of key genes involved in osteoclast function. To test this, primary osteoclasts were exposed to physiologically relevant concentrations of the prodrug tenofovir disoproxil fumarate (TDF), then intensive microarray analysis was done to compare tenofovir-treated versus untreated cells. Specific downregulation of Gnas, Got2 and Snord32a were observed in the TDF-treated cells. The functions of these genes help to explain the basis for tenofovir-associated bone density loss. Our studies represent the first analysis of the effects of tenofovir on osteoclast gene expression and help to explain the basis of tenofovir-associated bone density loss in HIV-infected individuals.
    Biochemical and Biophysical Research Communications 01/2010; · 2.28 Impact Factor
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    ABSTRACT: The uncoupling of osteoblastic and osteoclastic activity is central to disorders such as osteoporosis, osteolytic malignancies, and periodontitis. Numerous studies have shown explicit functions for bone morphogenetic proteins (BMPs) in skeletogenesis. Their signaling activity has been shown in various contexts to be regulated by extracellular proteins, including Twisted gastrulation (TWSG1). However, experimental paradigms determining the effects of BMP regulators on bone remodeling are limited. In this study, we assessed the role of TWSG1 in postnatal bone homeostasis. Twsg1-deficient (Twsg1(-/-)) mice developed osteopenia that could not be explained by defective osteoblast function, because mineral apposition rate and differentiation markers were not significantly different compared with wildtype (WT) mice. Instead, we discovered a striking enhancement of osteoclastogenesis in Twsg1(-/-) mice, leading to increased bone resorption with resultant osteopenia. Enhanced osteoclastogenesis in Twsg1(-/-) mice was caused by increased cell fusion, differentiation, and function of osteoclasts. Furthermore, RANKL-mediated osteoclastogenesis and phosphorylated Smad1/5/8 levels were enhanced when WT osteoclasts were treated with recombinant BMP2, suggesting direct regulation of osteoclast differentiation by BMPs. Increase in detectable levels of phosphorylated Smad 1/5/8 was noted in osteoclasts from Twsg1(-/-) mice compared with WT mice. Furthermore, the enhanced osteoclastogenesis in Twsg1(-/-) mice was reversed in vitro in a dose-dependent manner with exposure to Noggin, a BMP antagonist, strongly suggesting that the enhanced osteoclastogenesis in Twsg1 mutants is attributable to increased BMP signaling. Thus, we present a novel and previously uncharacterized role for TWSG1 in inhibiting osteoclastogenesis through regulation of BMP activity.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 06/2009; 24(11):1917-26. · 6.04 Impact Factor
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    ABSTRACT: We investigated the role of the alcohol environment in explaining disparities in homicide rates among minorities in 10 cities in the United States using 2003 data from the Malt Liquor and Homicide study. We hypothesized that (a) higher concentrations of African Americans would be associated with higher homicide rates, as well as higher alcohol and malt liquor availability and promotion, and (b) the relationship between neighborhood racial/ethnic concentration and homicide would be attenuated by the greater alcohol and malt liquor availability and promotion in African American neighborhoods. Hypotheses were tested using separate Poisson, linear, and logistic regression models that corrected for spatial autocorrelation. Census block groups served as the unit of analysis (n = 450). We found that higher concentrations of African Americans were associated with higher homicide rates as well as greater alcohol availability, especially malt liquor availability. The promotion of malt liquor on storefronts was also significantly greater in African American than in other neighborhoods. However, none of the measures representing alcohol or malt liquor availability and promotion variables changed the effect of neighborhood racial/ethnic concentration on homicide. Limitations and implications of our findings are discussed.
    Substance Use &amp Misuse 02/2008; 43(2):159-77. · 1.11 Impact Factor

Publication Stats

115 Citations
31.79 Total Impact Points

Institutions

  • 2011–2012
    • University of Minnesota Duluth
      Duluth, Minnesota, United States
    • Saint Mary's University of Minnesota
      Minneapolis, Minnesota, United States
    • University of Minnesota Twin Cities
      • Department of Developmental and Surgical Sciences
      Minneapolis, MN, United States