Archives

  • 2018-07
  • 2018-10
  • 2018-11
  • 2019-04
  • 2019-05
  • 2019-06
  • 2019-07
  • 2019-08
  • 2019-09
  • 2019-10
  • 2019-11
  • 2019-12
  • 2020-01
  • 2020-02
  • 2020-03
  • 2020-04
  • 2020-05
  • 2020-06
  • 2020-07
  • 2020-08
  • 2020-09
  • 2020-10
  • 2020-11
  • 2020-12
  • 2021-01
  • 2021-02
  • 2021-03
  • 2021-04
  • 2021-05
  • 2021-06
  • 2021-07
  • 2021-08
  • 2021-09
  • 2021-10
  • 2021-11
  • 2021-12
  • 2022-01
  • 2022-02
  • 2022-03
  • 2022-04
  • 2022-05
  • 2022-06
  • 2022-07
  • 2022-08
  • 2022-09
  • 2022-10
  • 2022-11
  • 2022-12
  • 2023-01
  • 2023-02
  • 2023-03
  • 2023-04
  • 2023-05
  • 2023-06
  • 2023-07
  • 2023-08
  • 2023-09
  • 2023-10
  • 2023-11
  • 2023-12
  • 2024-01
  • 2024-02
  • 2024-03
  • 2024-04
  • 2024-05
  • 2024-06
  • br Materials and methods br Results br

    2019-10-09


    Materials and methods
    Results
    Discussion The investigation of oxysterols is highly complex and challenging, arising not only from the large number of molecules in this family, but also due to their low abundance in biological systems compared to cholesterol and their susceptibility for autoxidation, forming the same metabolites ex vivo as present in vivo [1]. Moreover, a given oxysterol can be derived from different substrates or may be formed non-enzymatically by autoxidation. In addition, different enzymes can catalyze the conversion of one particular oxysterol. Thus, it is crucial to identify the enzymes responsible for the generation and degradation of a given oxysterol. The generation of 7β25OHC by oxoreduction of 7kC through 11β-HSD1 to 7βOHC and further by hydroxylation at position 25 through CH25H or CYP27A1 has recently been postulated [34]; however, experimental verification was lacking. Similarly, experimental evidence was missing for the hypothesized hydroxylation of 7kC to 7k25OHC by CH25H. The present study demonstrated for the first time the enzymatic formation of 7k25OHC from 7kC by CH25H and of 7β25OHC from 7k25OHC by 11β-HSD1 in vitro. Kinetic analysis using microsomal preparations indicated a very high substrate affinity of 11β-HSD1 for 7k25OHC with an estimated KMapp value in the lower nanomolar range. Importantly, compared to the so far reported kinetic parameters of 11β-HSD1-dependent oxoreduction reactions of endogenous substrates (cortisone and 11-DHC (KMapp ˜250-500 nM) [13,24], 7kC (KMapp ˜500 nM) [13] and 7oxoLCA (KMapp ˜1000 nM) [16]) displayed 7k25OHC an approximately 10–30 times higher substrate affinity with a similar Vmax value as for the oxoreduction of cortisone. Regarding 11β-HSD2, AZD6738 (KMapp ˜200 nM) and corticosterone (KMapp ˜5-10 nM) [24] showed a 2–3 times and a 50–100 times lower substrate affinity compared to 7β25OHC. Interestingly, whilst 11β-HSD2 clearly has higher affinities towards glucocorticoids than 11β-HSD1, the opposite was found to be true for 7-oxygenated 25OHC. Due to the stereospecific actions of 11β-HSDs, metabolizing exclusively 7k25OHC and 7β25OHC, the potent EBI2 ligand 7α25OHC is neither formed nor degraded by these enzymes. Nevertheless, although the 7α-hydroxylated form was found to be preferred over the 7β-hydroxylated metabolite for EBI2 activation, 7β25OHC was still able to trigger an EBI2 response, with EC50 values in the lower nanomolar range [7,8]. Moreover, in the present study only very low activity of the 7-oxo metabolite 7k25OHC could be detected towards EBI2 activation in calcium mobilization and no effect on migration of macrophages was found for the concentrations tested. Thus, a novel glucocorticoid-independent pre-receptor regulation mechanism by 11β-HSDs could be demonstrated. Interestingly, 7k25OHC was found in another context to activate the oncoprotein Smoothened (Smo) and thereby tune Hedgehog pathway response [35]. However, since 7β25OHC (and 7α25OHC) have not yet been examined for their ability to modulate Smo activity, this might represent a valuable target mechanism for a further pre-receptor regulation mechanism involving 11β-HSDs. The physiological concentrations of 7k25OHC and 7β25OHC in plasma and tissues have not yet been assessed. Average concentrations of free 7α25OHC in plasma of healthy individuals of approximately 0.1 ng/mL (˜0.2 nM) were reported [[36], [37], [38]], and similar circulating concentrations of 7k25OHC and 7β25OHC might exist. Nevertheless, in situations of oxidative stress and inflammation, an excessive and especially local accumulation of oxysterols can occur, as e.g. seen in atherosclerotic plaques, where 7kC levels of up to 10 μM can be detected [39]. Interestingly, the chronic overexpression of CH25H, and thus the production of 25OHC, has been described to promote the accumulation of cholesteryl ester, which leads further to foam cell formation [40]. Moreover, pronounced accumulation of 7kC, through non-enzymatic reactions or CYP7A1-dependet hydroxylation of 7-dehydrocholesterol, has been found as well in other disease states such as Smith-Lemli-Opitz syndrome, Niemann-Pick disease or cerebrotendinous xanthomatosis [[41], [42], [43]]. However, the involvement of 7k25OHC and 7β25OHC in these diseases has not yet been assessed, representing interesting targets for further research.