Date of Award

5-17-2015

Document Type

Honors Thesis

Department

Biology

First Advisor

Tiffany Frey

Language

English

Abstract

Mevalonate kinase deficiency (MKD) occurs in patients with mutations in the mevalonate kinase (MVK) gene, which is responsible for the phosphorylation of mevalonate in the mevalonate pathway. Patients with MKD exhibit a systemic inflammatory phenotype marked by recurrent episodes of disturbed cytokine production and fever attacks. The mevalonate pathway is responsible for the production of non-sterol isoprenoid compounds such as farnesyl-­ and geranylgeranylpyrophosphate (FPP and GGPP). FPP and GGPP are lipid moieties that are critical for the proper functioning of cellular molecules including the Ras, Rho, and Rab families of small GTP-­binding proteins. Phenotypes of MKD have been linked to GGPP depletion in monocytes and macrophages and are exacerbated in the presence of inflammatory stimuli such as lipopolysaccharide (LPS), a component of the outer membrane of Gram-­negative bacteria. The goal of this study was to compare the response of different cell types to LPS following isoprenoid depletion. MKD can be modeled in cell culture by treating cells with statins, a class of drugs that inhibit 3-­ hydroxy-­3-­methyl glutaryl coenzyme A (HMG-­CoA) reductase, the enzyme that acts directly upstream of mevalonate. When RAW 264.7 murine macrophages were treated with lovastatin and stimulated with LPS, expression of the pro-­inflammatory markers IL-­1β, TNF-­α, and CD14 increased. An inhibitor of geranylgeranyl transferase (GGTase) I had a similar effect as lovastatin on the expression of these markers. Since GGTase I is the enzyme responsible for the transfer of GGPP to Rho GTPases, we hypothesized that one or more members of this protein family are responsible for these effects. Treatment with lovastatin followed by stimulation with LPS led to increased activation of Rho, but no detection of Rac1 activation was present. In addition, an inhibitor of Rho protein function partially blocked the increased levels of IL-­1β and TNF-­α following lovastatin treatment, indicating that Rho protein activation is playing a role in these effects. In order to further examine the role of isoprenoid depletion in human monocytes and macrophages, monocytes were isolated from human blood by centrifugal elutriation and differentiated to monocyte-­derived macrophages (MDMs) by treatment with macrophage colony stimulating factor (M-­CSF). In contrast to the RAW 264.7 cells, IL-­1β mRNA levels show a suppressed increase in human monocytes following LPS stimulation in the presence of lovastatin and no changes in CD14 mRNA levels were observed in either human monocytes or MDMs. These findings indicate substantial cell specific differences in the response to LPS following isoprenoid depletion and require further investigation to determine the contribution of different cell types to the phenotypes of MKD. Ultimately, a better mechanistic understanding of these processes has the potential to impact treatment of inflammatory diseases such as MKD.

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