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Scholarly Works (32 results)

  • Thesis
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UC Merced Electronic Theses and Dissertations (2016)

Abstract

High-throughput Analysis of WNT Signaling Pathway in Osteoblasts

by

Aimy Sebastian

Doctor of Philosophy

University of California, Merced, 2016

Dr. Suzanne S. Sindi, Chair

Dr. David H. Ardell

Dr. Gabriela G. Loots

Dr. Jennifer O. Manilay

Multiple signaling pathways have been shown to regulate bone development and metabolism, and the WNT signaling pathway is emerging as one of the most crucial contributors. Several WNT ligands, receptors and WNT antagonists are expressed in bone and play a role in maintaining postnatal bone homeostasis. However, specific functions of individual WNT pathway members in bone are only beginning to be elucidated. Investigating the role of WNT signaling in bone development and metabolism will provide important implications for the treatment of fractures and bone thinning disorders such as osteoporosis and osteopenia. The focus of my thesis is to elucidate the functions of three out of nineteen WNT ligands and WNT co-receptors LRP5 and LRP6 in osteoblasts (bone forming cells).

In this thesis, I investigated the role of WNT ligands WNT3A, WNT5A and WNT16 in osteoblasts to identify the target genes regulated by these WNTs and to understand the molecular mechanism by which these WNTs regulate bone metabolism. Gene expression analysis of neonatal osteoblasts treated with recombinant WNTs identified more than 1000 genes regulated by WNT signaling in osteoblasts and suggested that WNT3A and WNT16 positively regulate early stages of osteoblast differentiation and inhibit osteoblast maturation/mineralization.

I also studied the role of WNT co-receptors LRP5 and LRP6 in mediating canonical WNT signaling. LRP5 and LRP6 are two WNT co-receptors that have been linked to bone development and metabolism. Both LRP5 and LRP6 are required for normal postnatal bone homeostasis. However, their specific roles are not well understood. To determine the roles of LRP5 and 6 in mediating canonical WNT signaling, osteoblasts lacking Lrp5, Lrp6 and both Lrp5 and 6 were treated with recombinant WNT3A. The RNA isolated from all WNT3A treated samples were sequenced and analyzed to identify genes regulated through LRP5 and LRP6 and genes that do not require LRP5/6 for WNT3A induced transcriptional regulation. This study revealed that LRP6 plays a dominant role in mediating WNT3A signaling in osteoblasts.

Canonical WNTs such as WNT3A regulate target gene expression by activating TCF/LEF family transcription factors. These transcription factors bind to promoters and/or enhancers of target genes to induce gene transcription. To identify direct targets of canonical WNT signaling, using ChIP-seq, I identified TCF/LEF binding sites near WNT3A targets. More than 80% WNT3A targets had TCF/LEF binding sites in their promoter and/or enhancers. This study also identified more than 500 putative WNT inducible enhancers in osteoblasts. A subset of predicted WNT inducible enhancers was validated experimentally to confirm WNT3A inducible enhancer activity.

My findings expand our current understanding of the role of WNT signaling pathway in regulating osteoblast differentiation and function, as well as contribute to the knowledge of the WNT signaling pathway itself. The WNT target genes identified in this study may be further explored for their therapeutic potential in treating osteoporosis and other bone disorders.

    Cover page: High-throughput Analysis of WNT Signaling Pathway in Osteoblasts
    • Article
    • Peer Reviewed
    UC Davis Previously Published Works (2015)
    Dynamic interaction between prostate cancer and the bone microenvironment is a major contributor to metastasis of prostate cancer to bone. In this study, we utilized an in vitro co-culture model of PC3 prostate cancer cells and osteoblasts followed by microarray based gene expression profiling to identify previously unrecognized prostate cancer-bone microenvironment interactions. Factors secreted by PC3 cells resulted in the up-regulation of many genes in osteoblasts associated with bone metabolism and cancer metastasis, including Mmp13, Il-6 and Tgfb2, and down-regulation of Wnt inhibitor Sost. To determine whether altered Sost expression in the bone microenvironment has an effect on prostate cancer metastasis, we co-cultured PC3 cells with Sost knockout (Sost(KO)) osteoblasts and wildtype (WT) osteoblasts and identified several genes differentially regulated between PC3-Sost(KO) osteoblast co-cultures and PC3-WT osteoblast co-cultures. Co-culturing PC3 cells with WT osteoblasts up-regulated cancer-associated long noncoding RNA (lncRNA) MALAT1 in PC3 cells. MALAT1 expression was further enhanced when PC3 cells were co-cultured with Sost(KO) osteoblasts and treatment with recombinant Sost down-regulated MALAT1 expression in these cells. Our results suggest that reduced Sost expression in the tumor microenvironment may promote bone metastasis by up-regulating MALAT1 in prostate cancer.
      Cover page: Cancer–Osteoblast Interaction Reduces Sost Expression in Osteoblasts and Up-Regulates lncRNA MALAT1 in Prostate Cancer
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2017)
      Wnt3a is a major regulator of bone metabolism however, very few of its target genes are known in bone. Wnt3a preferentially signals through transmembrane receptors Frizzled and co-receptors Lrp5/6 to activate the canonical signaling pathway. Previous studies have shown that the canonical Wnt co-receptors Lrp5 and Lrp6 also play an essential role in normal postnatal bone homeostasis, yet, very little is known about specific contributions by these co-receptors in Wnt3a-dependent signaling. We used high-throughput sequencing technology to identify target genes regulated by Wnt3a in osteoblasts and to elucidate the role of Lrp5 and Lrp6 in mediating Wnt3a signaling. Our study identified 782 genes regulated by Wnt3a in primary calvarial osteoblasts. Wnt3a up-regulated the expression of several key regulators of osteoblast proliferation/ early stages of differentiation while inhibiting genes expressed in later stages of osteoblastogenesis. We also found that Lrp6 is the key mediator of Wnt3a signaling in osteoblasts and Lrp5 played a less significant role in mediating Wnt3a signaling.
        Cover page: Wnt co-receptors Lrp5 and Lrp6 differentially mediate Wnt3a signaling in osteoblasts
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2021)
        Articular cartilage is a connective tissue lining the surfaces of synovial joints. When the cartilage severely wears down, it leads to osteoarthritis (OA), a debilitating disease that affects millions of people globally. The articular cartilage is composed of a dense extracellular matrix (ECM) with a sparse distribution of chondrocytes with varying morphology and potentially different functions. Elucidating the molecular and functional profiles of various chondrocyte subtypes and understanding the interplay between these chondrocyte subtypes and other cell types in the joint will greatly expand our understanding of joint biology and OA pathology. Although recent advances in high-throughput OMICS technologies have enabled molecular-level characterization of tissues and organs at an unprecedented resolution, thorough molecular profiling of articular chondrocytes has not yet been undertaken, which may be in part due to the technical difficulties in isolating chondrocytes from dense cartilage ECM. In this study, we profiled articular cartilage from healthy and injured mouse knee joints at a single-cell resolution and identified nine chondrocyte subtypes with distinct molecular profiles and injury-induced early molecular changes in these chondrocytes. We also compared mouse chondrocyte subpopulations to human chondrocytes and evaluated the extent of molecular similarity between mice and humans. This work expands our view of chondrocyte heterogeneity and rapid molecular changes in chondrocyte populations in response to joint trauma and highlights potential mechanisms that trigger cartilage degeneration.
          Cover page: Single-Cell RNA-Seq Reveals Transcriptomic Heterogeneity and Post-Traumatic Osteoarthritis-Associated Early Molecular Changes in Mouse Articular Chondrocytes
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2022)
          Type 1 diabetes mellitus (T1DM) affects 9.5% of the population. T1DM is characterized by severe insulin deficiency that causes hyperglycemia and leads to several systemic effects. T1DM has been suggested as a risk factor for articular cartilage damage and loss, which could expedite the development of osteoarthritis (OA). OA represents a major public health challenge by affecting 300 million people globally, yet very little is known about the correlation between T1DM and OA. In addition, current studies that have looked at the interaction between diabetes mellitus and OA have reported conflicting results with some suggesting a positive correlation whereas others did not. In this study, we aimed to evaluate whether T1DM exacerbates the development of spontaneous OA or accelerates the progression of posttraumatic osteoarthritis (PTOA) after joint injury. Histological evaluation of T1DM and control joints determined that T1DM mice displayed cartilage degeneration measurements consistent with mild OA phenotypes. RNA sequencing analyses identified significantly upregulated genes in T1DM corresponding to matrix-degrading enzymes known to promote cartilage matrix degradation, suggesting a role of these enzymes in OA development. Next, we assessed whether preexisting T1DM influences PTOA development subsequent to trauma. At 6 weeks post-injury, T1DM injured joints displayed significantly less cartilage damage and joint degeneration than injured non-diabetic joints, suggesting a significant delay in PTOA disease progression. At the single-cell resolution, we identified increased number of cells expressing the chondrocyte markers Col2a1, Acan, and Cytl1 in the T1DM injured group. Our findings demonstrate that T1DM can be a risk factor for OA but not for PTOA. This study provides the first account of single-cell resolution related to T1DM and the risk for OA and PTOA. © 2022 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.
            Cover page: Preexisting Type 1 Diabetes Mellitus Blunts the Development of Posttraumatic Osteoarthritis
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2017)
            Joint injury causes post-traumatic osteoarthritis (PTOA). About ∼50% of patients rupturing their anterior cruciate ligament (ACL) will develop PTOA within 1-2 decades of the injury, yet the mechanisms responsible for the development of PTOA after joint injury are not well understood. In this study, we examined whole joint gene expression by RNA sequencing (RNAseq) at 1 day, 1-, 6-, and 12 weeks post injury, in a non-invasive tibial compression (TC) overload mouse model of PTOA that mimics ACL rupture in humans. We identified 1446 genes differentially regulated between injured and contralateral joints. This includes known regulators of osteoarthritis such as MMP3, FN1, and COMP, and several new genes including Suco, Sorcs2, and Medag. We also identified 18 long noncoding RNAs that are differentially expressed in the injured joints. By comparing our data to gene expression data generated using the surgical destabilization of the medial meniscus (DMM) PTOA model, we identified several common genes and shared mechanisms. Our study highlights several differences between these two models and suggests that the TC model may be a more rapidly progressing model of PTOA. This study provides the first account of gene expression changes associated with PTOA development and progression in a TC model. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. J Orthop Res 35:474-485, 2017.
              Cover page: Global molecular changes in a tibial compression induced ACL rupture model of post‐traumatic osteoarthritis
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2020)
              Osteoarthritis (OA) is a painful and debilitating disease characterized by the chronic and progressive degradation of articular cartilage. Post-traumatic OA (PTOA) is a secondary form of OA that develops in ~50% of cases of severe articular injury. Inflammation and re-occurring injury have been implicated as contributing to the progression of PTOA after the initial injury. However, there is very little known about external factors prior to injury that could affect the risk of PTOA development. To examine how the gut microbiome affects PTOA development we used a chronic antibiotic treatment regimen starting at weaning for six weeks prior to ACL rupture, in mice. A six-weeks post-injury histological examination showed more robust cartilage staining on the antibiotic (AB)-treated mice than the untreated controls (VEH), suggesting slower disease progression in AB cohorts. Injured joints also showed an increase in the presence of anti-inflammatory M2 macrophages in the AB group. Molecularly, the phenotype correlated with a significantly lower expression of inflammatory genes Tlr5, Ccl8, Cxcl13, and Foxo6 in the injured joints of AB-treated animals. Our results indicate that a reduced state of inflammation at the time of injury and a lower expression of Wnt signaling modulatory protein, Rspo1, caused by AB treatment can slow down or improve PTOA outcomes.
                Cover page: Antibiotic Treatment Prior to Injury Improves Post-Traumatic Osteoarthritis Outcomes in Mice
                • Article
                • Peer Reviewed
                UC Davis Previously Published Works (2022)
                We previously showed that femur fracture in mice caused a reduction in bone volume at distant skeletal sites within 2 weeks post-fracture. Osteocytes also have the ability to remodel their surrounding bone matrix through perilacunar/canalicular remodeling (PLR). If PLR is altered systemically following fracture, this could affect bone mechanical properties and increase fracture risk at all skeletal sites. In this study, we investigated whether lacunar-canalicular microstructure and the rate of PLR are altered in the contralateral limb following femoral fracture in mice. We hypothesized that femoral fracture would accelerate PLR by 2 weeks postfracture, followed by partial recovery by 4 weeks. We used histological evaluation and high-resolution microcomputed tomography to quantify the morphology of the lacunar-canalicular network at the contralateral tibia, and we used quantitative real-time polymerase chain reaction (RT-PCR) and RNA-seq to measure the expression of PLR-associated genes in the contralateral femur. We found that at both 2 and 4 weeks postfracture, canalicular width was significantly increased by 18.6% and 16.6%, respectively, in fractured mice relative to unfractured controls. At 3 days and 4 weeks post-fracture, we observed downregulation of PLR-associated genes; RNA-seq analysis at 3 days post-fracture showed a deceleration of bone formation and mineralization in the contralateral limb. These data demonstrate notable canalicular changes following fracture that could affect bone mechanical properties. These findings expand our understanding of systemic effects of fracture and how biological and structural changes at distant skeletal sites may contribute to increased fracture risk following an acute injury.
                  Cover page: Altered canalicular remodeling associated with femur fracture in mice
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2022)
                  Fentanyl is one of the most common opioid analgesics administered to patients undergoing surgery or for chronic pain management. While the side effects of chronic fentanyl abuse are recognized (e.g., addiction, tolerance, impairment of cognitive functions, and inhibit nociception, arousal, and respiration), it remains poorly understood what and how changes in brain activity from chronic fentanyl use influences the respective behavioral outcome. Here, we examined the functional and molecular changes to cortical neural network activity following sub-chronic exposure to two fentanyl concentrations, a low (0.01 μM) and high (10 μM) dose. Primary rat co-cultures, containing cortical neurons, astrocytes, and oligodendrocyte precursor cells, were seeded in wells on either a 6-well multi-electrode array (MEA, for electrophysiology) or a 96-well tissue culture plate (for serial endpoint bulk RNA sequencing analysis). Once networks matured (at 28 days in vitro), co-cultures were treated with 0.01 or 10 μM of fentanyl for 4 days and monitored daily. Only high dose exposure to fentanyl resulted in a decline in features of spiking and bursting activity as early as 30 min post-exposure and sustained for 4 days in cultures. Transcriptomic analysis of the complex cultures after 4 days of fentanyl exposure revealed that both the low and high dose induced gene expression changes involved in synaptic transmission, inflammation, and organization of the extracellular matrix. Collectively, the findings of this in vitro study suggest that while neuroadaptive changes to neural network activity at a systems level was detected only at the high dose of fentanyl, transcriptomic changes were also detected at the low dose conditions, suggesting that fentanyl rapidly elicits changes in plasticity.
                    Cover page: Dose-dependent consequences of sub-chronic fentanyl exposure on neuron and glial co-cultures.
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2018)
                    Patients with anterior cruciate ligament (ACL) rupture are two times as likely to develop posttraumatic osteoarthritis (PTOA). Annually, there are ∼900,000 knee injuries in the United States, which account for ∼12% of all osteoarthritis (OA) cases. PTOA leads to reduced physical activity, deconditioning of the musculoskeletal system, and in severe cases requires joint replacement to restore function. Therefore, treatments that would prevent cartilage degradation post-injury would provide attractive alternatives to surgery. Sclerostin (Sost), a Wnt antagonist and a potent negative regulator of bone formation, has recently been implicated in regulating chondrocyte function in OA. To determine whether elevated levels of Sost play a protective role in PTOA, we examined the progression of OA using a noninvasive tibial compression overload model in SOST transgenic (SOSTTG ) and knockout (Sost-/- ) mice. Here we report that SOSTTG mice develop moderate OA and display significantly less advanced PTOA phenotype at 16 weeks post-injury compared with wild-type (WT) controls and Sost-/- . In addition, SOSTTG built ∼50% and ∼65% less osteophyte volume than WT and Sost-/- , respectively. Quantification of metalloproteinase (MMP) activity showed that SOSTTG had ∼2-fold less MMP activation than WT or Sost-/- , and this was supported by a significant reduction in MMP2/3 protein levels, suggesting that elevated levels of SOST inhibit the activity of proteolytic enzymes known to degrade articular cartilage matrix. Furthermore, intra-articular administration of recombinant Sost protein, immediately post-injury, also significantly decreased MMP activity levels relative to PBS-treated controls, and Sost activation in response to injury was TNFα and NF-κB dependent. These results provide in vivo evidence that sclerostin functions as a protective molecule immediately after joint injury to prevent cartilage degradation. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals Inc.
                      Cover page: SOST/Sclerostin Improves Posttraumatic Osteoarthritis and Inhibits MMP2/3 Expression After Injury
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