Vascular calcification greatly increases risk for heart disease, the leading cause of death in patients with diabetes and chronic kidney disease. Vascular calcification is an active process attributed to vascular smooth muscle cell (VSMC) mineralization. The sodium-dependent phosphate cotransporter, PiT-1, mediates VSMC calcification. The mechanisms by which PiT-1 causes calcification may include cellular matrix degradation, apoptosis, calcium phosphate vesicle release, and changes in gene expression. Of these mechanisms, my research focuses on the poorly understood signaling pathway by which PiT-1 induces VSMC calcification through up-regulating osteogenic (bone-forming) and chondrogenic (cartilage-forming) genes and down-regulating smooth muscle genes. These changes in gene expression make VSMCs more readily calcified in elevated phosphate. Recent literature suggests that VSMCs respond to elevated phosphate through PiT-1 with a novel cell signaling mechanism independent of phosphate uptake. My project concentrates on understanding the function of the PiT-1 protein's intracellular domain, which currently has no known function. I hypothesize that this section of PiT-1 is responsible for signaling in response to elevated phosphate, triggering a phosphorylation cascade that activates osteogenic transcription factors, such as Runx2, which initiate this osteogenic conversion. I am working toward integrating the gene for the intracellular domain of PiT-1 into the vascular smooth muscle cell genome to overexpress the truncated protein in cells. From my hypothesis that PiT-1 has a role in cell signaling, I predict that the overexpression of the intracellular domain of PiT-1 will reduce VSMC calcification by competing with the wild type PiT-1 protein for binding to proteins in the activation pathway, and inhibiting the signaling process. If my data support this hypothesis, I may engineer the intracellular domain of PiT-1 for therapeutic purposes.

It has been shown that the chemical nor-binaltorphimine (norBNI) inactivates the k-opioid receptor (KOR) for days or weeks in vivo, and also results in downstream activation of c-Jun N-terminal kinase (JNK). If JNK is blocked in vivo by SP610025 or by knocking out the JNK1 gene, norBNI inactivates the KOR for just hours. We hypothesize that there is a JNK mediated inactivation of the KOR. To further investigate this, I will create constructs that will allow us to study the interactions of JNK and arrestin, two proteins that are involved in the transduction of KOR signaling. I aim to express a version of each JNK1 splice variant tagged with luciferase and arrestin3 tagged with Venus in human embryonic kidney (HEK293) cells that have been transfected with rat KOR. Venus and luciferase are two bioluminescent molecules that emit different wavelengths of light depending on their distance from one another. After treating these cells with norBNI, I can dtermine the level of their interaction by using a bioluminescence resonance energy transfer (BRET) assay. These experiments will provide useful data in determining the extent of the interactions between JNK1 and arrestin3. This data will help us understand the protein cascade that results in the deactivation of the KOR, and if certain drugs activate certain splice variants. Understanding this pathway has implications in creating analgesics with smaller potential for abuse and addiction, and creating pharmaceuticals that could help with stress related depression and addiction relapse.

The aim of this study was to further the understanding of mechanisms that underlie the pruritic and antipruritic effects of kappa opioid receptor drugs. The kappa opioid receptor (KOR) has been studied for its potential therapeutic benefits. It has been previously shown that the kappa opioid receptor antagonist, 5’-guanidinonaltrindole (GNTI), causes compulsive, hind-leg scratching in mice, while the kappa opioid receptor agonist, nalfurafine, inhibits this pruritic effect. There are applications for this antipruritic effect in medicine, to counteract symptoms in kidney-failure patients. By learning more about the mechanisms of this anti-pruritic effect, better drugs can be developed in the future with increased specificity and decreased side effects. We pre-treated male C57/BL6 mice with either saline or nalfurafine (0.05mg/kg, s.c. flank), and challenged them 20 minutes later with 5’-GNTI (0.03mg/kg, s.c. behind the neck) or saline. The number of hind-leg scratches in the 30 minutes following GNTI injection were counted as the measure of itch. We compared the results between kappa opioid receptor (KOR) knock-out mice, mu opioid receptor (MOR) knock-out mice, and wild-type mice, to determine the role of KOR and MOR in GNTI-induced itch and nalfurafine inhibition of itch. We concluded that while the pruritic effects of 5’-GNTI are not mediated by KOR or MOR, the antipruritic effects of nalfurafine was KOR dependent.