@phdthesis{Schlereth2013,
  author    = {Schlereth, Florian},
  title     = {Expression of the DHEA/DHEAS-Shuttle in cell lines and foetal tissue of human liver, adrenal and cartilage},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-102068},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2013},
  abstract  = {DHEA is a precursor for the male and female sex hormones testosterone and estradiol, which are mainly secreted from the testes and the ovary, respectively. In addition, epidemiological studies showed that low serum levels of DHEA and DHEAS correlate with the incidence of autoimmune disease, cancer and cardiovascular disease. In vitro, DHEA and DHEAS influenced glucose metabolism in a favourable manner. However, positive effects of DHEA substitution were only significant adrenal insufficiency in women. Steroid sulphotransferase 2A1 (SULT2A1) is the responsible enzyme for sulphonation of DHEA to DHEAS which is thought to be the inactive form of DHEA. In this role, SULT2A1 acts as a central regulator of steroid synthesis because sulphonation of DHEA withdraws the substrate for further downstream conversion. Another essential cofactor for sulphonation is PAPS, which is produced by the enzyme PAPS synthase (PAPSS) from ATP and anorganic sulphate. PAPSS exists in the different isoforms PAPSS1 and PAPSS2 and splice variants PAPSS2a and PAPSS2b. Changes in PAPSS activity are thought to influence sulphonation of DHEA significantly. However, neither regulation of PAPSS nor its influence on SULT2A1 have been investigated in human cell lines or humans. The main goal of this thesis was to analyze the enzyme expression of the DHEA/DHEA shuttle, i.e. mRNA and protein of SULT2A1, PAPSS1 and PAPSS2, in various human cell lines. Furthermore, I investigated which cell line could serve as a suitable model for further research regarding regulation of SULT2A1, PAPSS1 and PAPSS2. Here, I could show that the enzymes of the DHEA/DHEAS shuttle were expressed in the human adrenal cell line NCI-h295R as both mRNA and protein. In enzyme assays, I was able to prove conversion of DHEA to DHEAS as well as to different other steroids. However, applying Trilostane, a potent inhibitor of CYP3B, effectively directed conversion of DHEA to DHEAS. Using these findings, future experiments can investigate for example the influence of certain cytokines or endocrine disruptors on expression and activity of PAPSS1/2 and on sulphonation of DHEA. In particular, the relatively equal expression of PAPSS1 and PAPSS2 will enable us to do knock down experiments with siRNA to elucidate how the activity of one enzyme changes when the other one fails. Sulphonation of DHEA by SULT2A1 is thought to happen in the cytoplasm or more precisely in the Golgi apparatus. However, experiments in transfected cells have shown both a cytoplasmatic and a nuclear localisation when both enzymes were expressed at the same time. Immunocytochemistry revealed the same results in the adrenal cell line NCI-h295R, where both enzymes were expressed strongly in the nucleus. The physiological role is not clear and requires further research. Presumably, sulphate is activated in the nucleus. However, one could also speculate that a shift of PAPSS to the nucleus could generate a reservoir, which can be activated by re-localisation to the cytoplasm when more PAPS is needed. Expression of SULT2A1 in some foetal tissues has been investigated earlier. Whilst in adult human cartilage PAPSS1 is predominant, in newly born hamsters PAPSS2 is more abundantly expressed. The expression of PAPSS isoforms in highly sulphonating tissue has not been investigated in humans, so far. This work demonstrated a differential expression of SULT2A1, PAPSS1 and PAPSS2 in adult and foetal liver, adrenal and foetal cartilage tissue. In adult and foetal adrenal expression was similar. However, foetal and adult liver differed in the expression of SULT2A1, which was expressed much more in adult tissue. Most importantly, in foetal cartilage there was only a low expression of SULT2A1 and PAPS seems to mostly provided by PAPSS1, which was considerably higher expressed in cartilage than in other tissues. In contrast, PAPSS2 was mainly expressed in adult and foetal adrenal. Additionally, we reported a case of a female patient who had been investigated for hyperandrogenism. Two mutations in the PAPSS2 gene had led to massively reduced serum levels of DHEAS. One heterozygous mutation in the domain of the APS kinase of the PAPSS2 protein leads to substitution of one amino acid at position 48 (T48R). In vitro experiments showed a residual activity of 6\% for this mutation. A second mutation in the ATP sulphurylase domain of PAPSS2 was found. The introduction of thymidine instead of cytidine leads to a stop codon, which is presumed to truncate the protein at position 329 (R329X). In vitro, no residual activity was seen for this mutation. The lack of PAPS reduces sulphonation of DHEA but also sulphonation of proteoglycanes, which leads to skeletal abnormalities. The abundance of DHEA enables massive downstream conversion to androgens leading to clinical features of hyperandrogenism. Regarding the bone abnormalities, it is interesting and surprising that activity of PAPSS1 compensated to a great extent in cartilage but was not able to keep up a more considerable sulphonation of DHEA. Possibly, the subcellular localisation might play a role in this scenario.},
  subject      = {Dehydroepiandrosteron},
  language  = {en}
}