Lysophosphatidic acid (LPA) is the simplest of the glycerol lipids and regulates a number of cellular processes such as morphological changes, migration, proliferation, and inhibition of apoptosis. LPA exerts these effects through activation of the G-protein coupled receptors (GPCRs) LPA1-6 and the intracellular fatty acid receptor peroxisome proliferator-activated receptor-gamma (PPARγ). The overall goal of this thesis was to determine the mechanisms by which LPA enhances cell survival by inhibiting apoptosis. The project was divided into three studies: 1) to determine the mechanism of LPA-mediated inhibition of p53 in A549 lung carcinoma cells, 2) to investigate the regulation of growth plate chondrocytes by LPA, and 3) to determine the mechanisms of LPA-mediated effects in the growth plate. In the first study, evidence is provided that LPA reduces the cellular abundance of the tumor suppressor p53 in A549 lung carcinoma cells. The LPA effect depends upon increased proteasomal degradation of p53 and it results in a corresponding decrease in p53-mediated transcription. The result of LPA-mediated inhibition of p53 in A549 cells is enhanced resistance to chemotherapeutic-induced apoptosis. In the second study, the role of LPA in resting zone chondrocytes (RC cells) was investigated. RC cells are regulated by 24,25-dihydroxyvitamin D3 [24,25(OH)[subscript2]D [subscript 3]] via a phospholipase D-dependent pathway, suggesting downstream phospholipid metabolites are involved. In this study, we showed that 24R,25(OH)[subscript 2]D[subscript 3] stimulates rat costochondral RC cells to release LPA. Additionally, we demonstrated that RC cells respond to LPA with increased proliferation, maturation, and inhibition of apoptosis. In the final study, the mechanism of LPA and 24R,25(OH)[subscript 2]D[subscript 3]-mediated inhibition of chondrocyte apoptosis was further investigated. Our data show that 24R,25(OH)[subscript 2]D[subscript 3] inhibits apoptosis through Ca⁺⁺, PLD, and PLC signaling and through LPA/Gαi/PI[subscript 3]K/mdm2-mediated degradation of p53, resulting in decreased caspase-3 activity. Collectively, our data establish LPA, vitamin D, and p53 as an anti-apoptotic signaling axis.

LPA1 lysophosphatidic acid receptors (LPA1Rs) are normally present on the surface of the cell. Our initial findings were that HMG-CoA reductase inhibitors (atorvastatin and mevastatin) induce the sequestration of the G protein-coupled LPA1R in recycling endosomes, most likely by inhibiting the recycling of tonically internalized receptors. Whereas, co-addition of geranylgeranylpyrophosphate (GGPP) or geranylgeraniol (GGOH) prevented atorvastatin-induced sequestration of LPA1Rs, the geranylgeranyltransferase-I inhibitor, GGTI-298, mimicked atorvastatin and induced LPA1R sequestration. This suggested that statin-induced endosomal sequestration was caused by defective protein prenylation. The likely targets of atorvastatin and GGTI-298 are the Rho family GTPases, RhoC and RhoA, since both inhibitors greatly reduced the abundance of these GTPases and since knockdown of endogenous RhoC or RhoA with small interfering RNAs (siRNAs) led to endosomal sequestration of LPA1R. Knockdown of RhoC was much more potent at inducing endosomal sequestration than knockdown of either RhoA or RhoB. In contrast, atorvastatin, GGTI-298, siRNA against RhoA, B, or C did not alter the internalization or recycling of transferrin receptors, indicating that recycling of transferrin receptors is distinct from LPA1Rs. Thus, these results, for the first time, implicate RhoA and RhoC in endocytic recycling of LPA1Rs and identify atorvastatin and GGTI-298 as novel inhibitors of this process. / Per the request of the author and advisor, and with the approval of the Graduate Education office, the following changes were made to this thesis: Replaced original page 1 with Errata Page 2. Replaced original pages 3-28 with Errata Pages 3 – 16. Replaced original pages 69-71 with Errata pages 17 – 19.

INTRODUCTION: The blood brain barrier (BBB), formed by the brain capillary endothelial cells separating the blood from the brain. Furthermore, the brain endothelial cells also express numerous transporter systems which help regulate and maintain the brain microenvironment. The protective function of the BBB and their transporter systems under pathological disease states, including brain tumor, can be an obstacle for the entry of therapeutic agents to the brain. OBJECTIVES: The current study set out to characterize brain tumor-induced alterations of the BBB of a mouse brain tumor model. Studies were performed to address changes in BBB permeability to P-gp dependent solutes using Rhodamine (R800). Furthermore, the use of lysophosphatidic acid (LPA) to modulate BBB permeability was also examined in healthy mice and tumor-bearing mice. METHODS: Tumors were induced by injecting Lewis Lung carcinoma (3LL) cells into the right hemisphere of female Balb/c mice. Changes in BBB permeability were assessed at various stages of tumor development, using both gadolinium contrast-enhanced agent (Gad) and 3H-mannitol. Functional activity of P-gp in the BBB was examined in adult mice following i.v. injection of R800 in the presence and absence of GF120918 (a P-gp inhibitor). Alterations in BBB permeability were characterized in healthy and tumor-bearing mice using a small (Gad) and large (IRdye800cw PEG) vascular permeability agent as well as R800 (changes in P-gp mediated permeability). RESULTS: Median mouse survival following 3LL injection was 17 days. The BBB was largely intact during tumor development with disruptions observed at the later stages of tumor development as indicated by Gad permeability. By inhibiting the function of P-gp with GF120918, the distribution of R800 in the brain increased by 4-fold. The enhancement effect of LPA on BBB permeability occurs within 3-6 minutes of injection with the barrier being restored back to its normal function within 20 minutes. Furthermore, an increased in brain penetration of IRdye800ce PEG and R800 were observed following LPA injection in both healthy and tumo-bearing mice. CONCLUSION: These studies provide the initial proof of concept for the use of BBB modulators including LPA and GF120918 to enhance drug delivery to the brain and the tumor sites.

INTRODUCTION: The blood brain barrier (BBB), formed by the brain capillary endothelial cells separating the blood from the brain. Furthermore, the brain endothelial cells also express numerous transporter systems which help regulate and maintain the brain microenvironment. The protective function of the BBB and their transporter systems under pathological disease states, including brain tumor, can be an obstacle for the entry of therapeutic agents to the brain. OBJECTIVES: The current study set out to characterize brain tumor-induced alterations of the BBB of a mouse brain tumor model. Studies were performed to address changes in BBB permeability to P-gp dependent solutes using Rhodamine (R800). Furthermore, the use of lysophosphatidic acid (LPA) to modulate BBB permeability was also examined in healthy mice and tumor-bearing mice. METHODS: Tumors were induced by injecting Lewis Lung carcinoma (3LL) cells into the right hemisphere of female Balb/c mice. Changes in BBB permeability were assessed at various stages of tumor development, using both gadolinium contrast-enhanced agent (Gad) and 3H-mannitol. Functional activity of P-gp in the BBB was examined in adult mice following i.v. injection of R800 in the presence and absence of GF120918 (a P-gp inhibitor). Alterations in BBB permeability were characterized in healthy and tumor-bearing mice using a small (Gad) and large (IRdye800cw PEG) vascular permeability agent as well as R800 (changes in P-gp mediated permeability). RESULTS: Median mouse survival following 3LL injection was 17 days. The BBB was largely intact during tumor development with disruptions observed at the later stages of tumor development as indicated by Gad permeability. By inhibiting the function of P-gp with GF120918, the distribution of R800 in the brain increased by 4-fold. The enhancement effect of LPA on BBB permeability occurs within 3-6 minutes of injection with the barrier being restored back to its normal function within 20 minutes. Furthermore, an increased in brain penetration of IRdye800ce PEG and R800 were observed following LPA injection in both healthy and tumo-bearing mice. CONCLUSION: These studies provide the initial proof of concept for the use of BBB modulators including LPA and GF120918 to enhance drug delivery to the brain and the tumor sites.

Addition of lysophosphatidic acid (LPA) to rat aorta-derived endothelial cells significantly induced preproendothelin-1 (preproET-1) mRNA expression. PreproET-1 mRNA levels reached a plateau within 1 h after the addition of 0.5 μM LPA and declined after 2 h. The induction was superinduced by cycloheximide and was blocked by actinomycin D. Suramin, an LPA receptor antagonist, abolished the induction of preproET-1 mRNA by LPA. Protein kinase C inhibitors, H7 and bisindolylmaleimide, were able to block the induction. Transient transfection experiment revealed that the elevated preproET-1 mRNA was a result of the activation of ET-1 gene activity. Electrophoretic mobility shift assay revealed that LPA stimulated the binding of AP-1. The secreted level of ET-1 was elevated 2.3-fold after 12 h of stimulation with LPA. Our results suggest that the upregulation of preproET-1 by LPA may serve to augment and prolong the vasoconstriction action of LPA.

No description available.

Ductal adenocarcinomas of the pancreas are the 4th most common cause of cancer death. The 1 and 5 year survival rates for all stages combined are currently 26% and 5% respectively. Median survival is less than 6 months. Despite remarkable progress in the fields of genetics, cancer biology, and advances in surgical techniques as well as chemotherapeutics, our ability to recognize and treat patients with pancreatic cancer remains poor. GPCR signaling modules have been increasingly implicated in the genesis and progression of pancreatic cancers. Aberrant agonist production, receptor expression and dysfunctional signaling resulting from genomic instability in a background of a heterotopic tumor-stromal microenvironment, contribute to the initiation, progression, and eventual metastasis of the disease. Numerous GPCR agonists, including lysophosphatidic acid (LPA), along with their cognate receptors have been implicated in this oncogenic process. LPA, one of the simplest bioactive lipids, has been shown to be a potent stimulant of metastatic behavior in in vitro models. It also acts as a mitogen by inducing proliferation and cell survival pathways in various normal and transformed cell lines. In patients with pancreatic cancer both the receptors and ligand have been found to be overexpressed. It has been noted that pancreatic cancer cell lines expressing higher levels of the LPA receptors present with greater motility. This has led to the hypothesis that LPA contributes to the progression of pancreatic cancer through the promotion of a metastatic phenotype. However, the underlying mechanisms have not been well described. LPA receptors have been shown to couple to the Gi, Gq, or G12 family of heterotrimeric G proteins. Consequently, signals transduced through these receptors have been shown to stimulate Gαi, Gαq, and Gα12/13 dependent pathways. While earlier studies have linked Gαi to LPA induced migration, there is recent evidence to suggest that Gα13 may provide a major signaling mechanism for LPA receptors stimulating migration in diverse cell types including cancer cell lines. Given the ominous nature of pancreatic cancers it is of critical importance to understand the mechanisms that promote more malignant phenotypes and to assess the role of Gα13 in this process. The goal of this thesis therefore is to define the role of Gα13 in LPA-mediated migration of pancreatic cancer cells. To assess the oncogenic potential of LPA and the role of Gα13 in stimulating the migration of pancreatic cancer cells, a panel of pancreatic cancer cell lines was assembled and characterized with regard to their expression of the LPA receptors as well as the Gα subunits of the heterotrimeric G proteins. These cell lines were further studied through a series of proliferation, wound healing, and transwell migration assays to assess the role of LPA in the induction of proliferation and migration in pancreatic cancer cells. The results demonstrated that LPA functions as a mitogen in certain pancreatic cancer cell lines, but is a potent stimulant of cell motility and invasive migration. Interestingly, these studies indicated that this response proceeds through routes that may not involve Gαi, as a potent migratory response was observed in MDAPanc28 cells which lack expression of the Gαi subunit. This was verified through the transwell assays conducted in the presence of PTX demonstrating that migration occurs independently of PTX sensitive mechanism and thus independently of Gαi.. Using a dominant negative mutant strategy, the studies presented in this thesis establishes the role of Gα13 in mediating LPA-LPAR stimulated migration of pancreatic cancer cells. Using pancreatic cancer cell lines that stably express the competitively inhibitory dominant negative mutant of Gα13, the ability of these mutants to inhibit a LPA mediated migratory response was monitored by wound-healing as well as transwell migration assays The results of these studies indicated a substantial attenuation of the migratory response and demonstrated for the first time the critical role of Gα13in LPA induced migration in a pancreatic cancer cell line. / Molecular Biology and Genetics

Ovarian cancer is currently the most fatal gynecologic cancer and the fifth leading cause of fatal cancer in women overall. As compared to the better-characterized malignancies, such as such as prostate, breast and colorectal cancers, there have been no major changes in methods of detection or treatment of ovarian cancers since the 1970's. As a result, the incidence and age-adjusted death rates for this disease have improved only marginally since that time. The molecular changes required for ovarian cancer pathogenesis remain poorly defined. Lysophosphatidic acid (LPA) has emerged as a biomarker present in the ascitic fluid and serum of ovarian cancer patients. Subsequent studies have identified LPA as an agonist for G protein coupled receptors (GPCRs). LPA has been well characterized as a pro-migratory factor in ovarian cancer and other cell systems. However, the role of LPA in mediating a proliferative response in ovarian cancer cells has yet to be fully characterized. In addition, the identity of the G protein pathways involved in this proliferative response remains a major unresolved question in the field. To investigate the mitogenic role of LPA in ovarian cancers, a panel of representative human ovarian cancer cells was assembled. A series of immunoblot and RT-PCR analyses was used to profile the LPA receptors and Gα-subunits expressed in these cells. In addition to verifying the migratory effect of LPA in these cells, a series of proliferation assays were used to investigate the potential role for LPA as a mitogen. The results indicate that stimulation with LPA results in a robust and statistically significant proliferative response. This response was quantified using multiple approaches. In addition, the proliferative response was observed in three independent ovarian cancer cell lines using concentrations of LPA within the range found in vivo in the ascitic fluid of ovarian cancer patients. Taken together, these data for the first time validate the role of LPA as a mitogen in ovarian cancer cells. To gain further insight into the oncogenic signaling response stimulated by LPA, activation of the mitogen activated protein kinase (MAPK) modules was determined. Using a series of immunoblot analyses and kinase assays, LPA was found to stimulate ERK as well as JNK modules. To investigate the functional roles of these pathways, a series of proliferation assays were carried out using inhibitors of ERK and JNK signaling. Consistent with the role of ERK as a crucial regulator of growth-factor induced proliferation in other cell systems, the results demonstrated a significantly attenuated growth response to LPA with ERK inhibition. Moreover, additional studies demonstrated for the first time that inhibition of JNK signaling significantly attenuates the proliferative response to LPA. In order to investigate the potential role of Gα12 in mediating the oncogenic response to LPA, the activation status of Gα12 was monitored in ovarian cancer cells stimulated with LPA. These studies demonstrate rapid activation of Gα12 with LPA stimulation. Finally to investigate the functional role of LPA-Gα12 signaling, a series of cell lines was established which express a dominant negative form of Gα12. Expression of this construct induced complete inhibition of Gα12 activation by LPA. These cells were then used to determine the effects of Gα12 inhibition on the oncogenic response to LPA. Consistent with the role of G12 family members in mediating cell migration, these cells demonstrated an attenuated migratory response to LPA. In addition, inhibition of Gα12 resulted in an attenuated proliferative response to serum. Finally, to investigate the role of Gα12 in mediating the proliferative response to LPA, a series of proliferation assays was carried out. The results indicated a significant > 50% inhibition in multiple ovarian cancer cell lines. Taken together, these results, presented here for the first time, establish that LPA is a potent mitogen that induces a proliferative response in human ovarian carcinoma cells. Although LPA had previously been shown to induce a proliferative response in multiple other cell types, it had not been known if LPA activates specific oncogenic pathways. This thesis tested the hypothesis that LPA, which is crucially involved in the pathophysiology of ovarian carcinoma, induces the activation of Gα12. In this context, the data presented here demonstrating a novel role for Gα12- which has been defined as the gep oncogene - in mediating this proliferative response in ovarian carcinoma, represents a major finding in the field. / Molecular Biology and Genetics

No / Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μM LPA in 60 μM Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mM Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway.

BACKGROUND:Lysophosphatidic acid (LPA) is a bioactive phospholipid with a potentially causative role in neurotrauma. Blocking LPA signaling with the LPA-directed monoclonal antibody B3/Lpathomab is neuroprotective in the mouse spinal cord following injury.FINDINGS:Here we investigated the use of this agent in treatment of secondary brain damage consequent to traumatic brain injury (TBI). LPA was elevated in cerebrospinal fluid (CSF) of patients with TBI compared to controls. LPA levels were also elevated in a mouse controlled cortical impact (CCI) model of TBI and B3 significantly reduced lesion volume by both histological and MRI assessments. Diminished tissue damage coincided with lower brain IL-6 levels and improvement in functional outcomes.CONCLUSIONS:This study presents a novel therapeutic approach for the treatment of TBI by blocking extracellular LPA signaling to minimize secondary brain damage and neurological dysfunction.