Our conclusion is in disagreement with that of a
Our conclusion is in disagreement with that of a recent paper by Park et al. , which suggests that nitric oxide (NO) inhibition of VHL recruitment occurs by nitrosylation of the 520 cysteine in HIF-1α, and that SNAP does not directly inhibit hydroxylation of HIF peptide by HPH-2. Although this discrepancy seems difficult to reconcile, it is possible that the truncated recombinant HPH-2 (amino acids 184–418) used in that paper may be less susceptible to NO than a full length HPH-2. Unlike a full length intact HPH-2 that shows much less activity in the absence of ferrous iron , the truncated HPH-2 was not affected by iron concentration, which raises a possibility that the catalytic activity (involving iron) and/or Km value (for dioxygen) of the truncated HPH-2 could also be altered from those of a full length HPH-2.
Our data suggest that NO inhibition of HPH-2 occurred by its competition with dioxygen binding. This argument is based on the observations that hyperoxia significantly attenuated the NO-mediated HIF-1α induction and restored VHL binding suppressed by NO donors. The specificity of the hyperoxia effect was evident in that hyperoxia had no effect on an iron chelator- or CAPE-mediated induction of HIF-1α and suppression of VHL binding. Moreover, hyperoxia-mediated HIF-1α degradation seems to occur by reactivation of HPHs, because the level of hydroxylated HIF-1α of the NO-donor treated now was much higher under hyperoxia than under normoxia in the presence of a proteasome inhibitor MG132 (Fig. 2C, lanes 6 vs 3).
Although SNAP effectively inhibited HPH-2, it did not impair CPHs activity as shown in Fig. 3. On the contrary, the known HPH inhibitors, ciclopirox and CAPE, drastically decreased the collagen hydroxyproline level. At high concentrations (>0.5mM), SNAP was found to elevate the collagen hydroxyproline level. Dooley et al. reported that another NO donor, DETA-NO, lowers activity of prolyl-4 hydroxylase to 76% of the control in fibroblasts , and suggested that this NO donor reduces collagen synthesis by impairing proline hydroxylation. On the other hand, there is a contrasting report that NO has a stimulatory effect on collagen synthesis in rat dermal fibroblasts , consistent with our findings. The discrepancy in NO effects on CPHs and collagen synthesis may be due to unknown variables in the different biological systems.
The two different prolyl-4 hydroxylases, HPHs and CPHs, have different Km values for dioxygen, and HPHs have much lower affinity for dioxygen. Consistent with this difference in kinetic parameters, HPHs display high sensitivity to hypoxic conditions, whereas CPHs seem to be fully functional under hypoxia , . HPHs are able to elicit their maximal activity under normoxia (21% oxygen) but lose their enzymatic activity as the oxygen concentration decreases, conferring their function as oxygen sensors . Considering these facts, selective inhibition of HPH-2 by NO competition of dioxygen binding may provide a rational explanation for the discriminative effect of NO toward CPHs and HPH-2. Thus, NO donors might be applicable for the treatment of diseases such as ischemic disorders in which HIF-1 activation is beneficial, without side effects resulting from the inhibition of CPHs.
Acknowledgements This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (No: 2009-0071594) and a grant of the Korea Healthcare technology R and D Project (A080640), Ministry for Health, Welfare and Family Affairs, Republic of Korea.
Introduction Collagen is one of the biodegradable materials. Historically, collagen has been already used in the field of leather, food, pharmaceutical applications, etc. For the past decade, collagen and its hydrolysate, gelatin, have been applied to cosmetics, and medical cosmetology, photographic industry. Collagen also has been utilized in tissue engineering scaffolds since 1980s. And collagen scaffolds are mainly for use as artificial skin, artificial bone, artificial tendon and artificial cartilage, etc. [, , ]. Recently controlled release drug delivery system is getting attention. Collagen and gelatin have been used as carriers for drug or growth factor in the form of membrane, sponge, gel, microsphere, injection and so on. Compared to other biodegradable natural polymers (chitosan, elastin, glycosaminoglycan, silk fibroin, etc.) and synthetic polymers ((polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), etc. ), collagen is the most significant constitutive protein, which plays a crucial role in the formation of the fundamental building block of connective tissues and its metabolism is associated with many physiological process of biological adaptations and tissue regeneration. In addition, collagen also has its superiority (abundant source, biocompatibility, easy processing, hydrophilicity, low antigenicity, absorbability in the body, etc.). But it also suffers from some poor physicochemical properties, such as thermostability, mechanical strength and resistance to enzyme.