Heparan sulfate selectively inhibits the collagenase activity of cathepsin K.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in resorption of bone matrix. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS regulates the biological functions of CtsK, remains largely unknown. In this report, we discovered that HS is a multifaceted regulator of the structure and function of CtsK. Structurally, HS forms a highly stable complex with CtsK and induces its dimerization. Co-crystal structures of CtsK with bound HS oligosaccharides reveal the location of the HS binding site and suggest how HS may support dimerization. Functionally, HS plays a dual role in regulating the enzymatic activity of CtsK. While it preserves the peptidase activity of CtsK by stabilizing its active conformation, it inhibits the collagenase activity of CtsK in a sulfation level-dependent manner. These opposing effects can be explained by our finding that the HS binding site is remote from the active site, which allows HS to specifically inhibit the collagenase activity without affecting the peptidase activity. At last, we show that structurally defined HS oligosaccharides effectively block osteoclast resorption of bone in vitro without inhibiting osteoclast differentiation, which suggests that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor for many diseases involving exaggerated bone resorption.

Heparan sulfate promotes TRAIL-induced tumor cell apoptosis.

TRAIL (TNF-related apoptosis-inducing ligand) is a potent inducer of tumor cell apoptosis through TRAIL receptors. While it has been previously pursued as a potential anti-tumor therapy, the enthusiasm subsided due to unsuccessful clinical trials and the fact that many tumors are resistant to TRAIL. In this report, we identified heparan sulfate (HS) as an important regulator of TRAIL-induced apoptosis. TRAIL binds HS with high affinity (KD = 73 nM) and HS induces TRAIL to form higher-order oligomers. The HS-binding site of TRAIL is located at the N-terminus of soluble TRAIL, which includes three basic residues. Binding to cell surface HS plays an essential role in promoting the apoptotic activity of TRAIL in both breast cancer and myeloma cells, and this promoting effect can be blocked by heparin, which is commonly administered to cancer patients. We also quantified HS content in several lines of myeloma cells and found that the cell line showing the most resistance to TRAIL has the least expression of HS, which suggests that HS expression in tumor cells could play a role in regulating sensitivity towards TRAIL. We also discovered that death receptor 5 (DR5), TRAIL, and HS can form a ternary complex and that cell surface HS plays an active role in promoting TRAIL-induced cellular internalization of DR5. Combined, our study suggests that TRAIL-HS interactions could play multiple roles in regulating the apoptotic potency of TRAIL and might be an important point of consideration when designing future TRAIL-based anti-tumor therapy.

Heparan sulfate selectively inhibits the collagenase activity of cathepsin K.

Cathepsin K (CtsK) is a cysteine protease with potent collagenase activity. CtsK is highly expressed by bone-resorbing osteoclasts and plays an essential role in bone remodeling. Although CtsK is known to bind heparan sulfate (HS), the structural details of the interaction, and how HS ultimately regulates the biological functions of CtsK, remains largely unknown. In this report, we determined that CtsK preferably binds to larger HS oligosaccharides, such as dodecasaccharides (12mer), and that the12mer can induce monomeric CtsK to form a stable dimer in solution. Interestingly, while HS has no effect on the peptidase activity of CtsK, it greatly inhibits the collagenase activity of CtsK in a manner dependent on sulfation level. By forming a complex with CtsK, HS was able to preserve the full peptidase activity of CtsK for prolonged periods, likely by stabilizing its active conformation. Crystal structures of Ctsk with a bound 12mer, alone and in the presence of the endogenous inhibitor cystatin-C reveal the location of HS binding is remote from the active site. Mutagenesis based on these complex structures identified 6 basic residues of Ctsk that play essential roles in mediating HS-binding. At last, we show that HS 12mers can effectively block osteoclast resorption of bone in vitro. Combined, we have shown that HS can function as a multifaceted regulator of CtsK and that HS-based oligosaccharide might be explored as a new class of selective CtsK inhibitor in many diseases that involve exaggerated bone resorption.

Heparan sulfate promotes TRAIL-induced tumor cell apoptosis.

TRAIL (TNF-related apoptosis-inducing ligand) is a potent inducer of tumor cell apoptosis through TRAIL receptors. While it has been previously pursued as a potential anti-tumor therapy, the enthusiasm subsided due to unsuccessful clinical trials and the fact that many tumors are resistant to TRAIL. In this report we identified heparan sulfate (HS) as an important regulator of TRAIL-induced apoptosis. TRAIL binds HS with high affinity (KD = 73 nM) and HS induces TRAIL to form higher-order oligomers. The HS-binding site of TRAIL is located at the N-terminus of soluble TRAIL, which includes three basic residues. Binding to cell surface HS plays an essential role in promoting the apoptotic activity of TRAIL in both breast cancer and myeloma cells, and this promoting effect can be blocked by heparin, which is commonly administered to cancer patients. We also quantified HS content in several lines of myeloma cells and found that the cell line showing the most resistance to TRAIL has the least expression of HS, which suggests that HS expression in tumor cells could play a role in regulating sensitivity towards TRAIL. We also discovered that death receptor 5 (DR5), TRAIL and HS can form a ternary complex and that cell surface HS plays an active role in promoting TRAIL-induced cellular internalization of DR5. Combined, our study suggests that TRAIL-HS interactions could play multiple roles in regulating the apoptotic potency of TRAIL and might be an important point of consideration when designing future TRAIL-based anti-tumor therapy.

Comparative evaluation of the morphological accuracy of dental crowns fabricated by different technologies.

STATEMENT OF PROBLEM: Information on the morphological accuracy of crowns produced by different technologies is limited. PURPOSE: The purpose of this in vitro study was to compare the morphology and contacts of crowns fabricated with intraoral systems, extraoral systems, and conventional method. MATERIAL AND METHODS: A typodont mandibular first molar (Nissin Dental Product) received a complete ceramic crown preparation and a reference crown. Microcomputed tomography (µCT) was used to obtain the virtual data (REF) of the reference crown. Three groups of replicated crowns were made: intraoral scanning system (TRIOS), extraoral scanning system (D700), and the conventional method (CONV) (n=8). The groups TRIOS and D700 were designed by the correlation method. All crowns were scanned with µCT to obtain 3D data. The data were superimposed on each other or the REF in an inspection software to evaluate precision and trueness. The contact penetration area of the occlusal surfaces of the crowns was calculated. An independent sample t test and 1-way ANOVA with the post hoc least significant difference (LSD) test were used to compare the data (α=.05). RESULTS: The crowns fabricated with the extraoral scanners showed significantly lower root mean square (RMS) values for trueness (F=1456.90, df=2, P<.001) and precision (F=188.88, df=2, P<.001) than the others. The penetration contact area ratio and the differences in the CONV group were both significantly higher than those of the other groups. CONCLUSIONS: The average discrepancies of the crown morphology fabricated from the extraoral scanning were significantly lower than those from others. The conventional method restored the occlusal contact with significantly less accuracy than the other groups.

Cyclic compression emerged dual effects on the osteogenic and osteoclastic status of LPS-induced inflammatory human periodontal ligament cells according to loading force.

BACKGROUND: Appropriate mechanical stimulation is essential for bone homeostasis in healthy periodontal tissues. While the osteogenesis and osteoclast differentiation of inflammatory periodontal ligament cells under different dynamic loading has not been yet clear. The aim of this study is to clarify the inflammatory, osteogenic and pro-osteoclastic effects of different cyclic stress loading on the inflammatory human periodontal ligament cells (hPDLCs). METHODS: hPDLCs were isolated from healthy premolars and cultured in alpha minimum Eagle's medium (α-MEM). Lipopolysaccharides (LPS) were used to induce the inflammation state of hPDLCs in vitro. Determination of LPS concentration for the model of inflammatory periodontium was based on MTT and genes expression analysis. Then the cyclic stress of 0, 0-50, 0-90 and 0-150 kPa was applied to the inflammatory hPDLCs for 5 days respectively. mRNA and protein levels of osteogenic, osteoclastic and inflammation-related markers were examined after the treatment. RESULTS: MTT and RT-PCR results showed that 10 µg/ml LPS up-regulated TNF-α, IL-1ß, IL-6, IL-8 and MCP-1 mRNA levels (P < 0.05) and did not affect the cell viability (P > 0.05). The excessive loading of stress (150 kPa) with or without LPS strongly increased the expression of inflammatory-related markers TNF-α, IL-1ß, IL-6, IL-8, MCP-1 (P < 0.05) and osteoclastic markers RANKL, M-CSF, PTHLH and CTSK compared with other groups (P < 0.05), but had no significant effect on osteogenic genes. While 0-90 kPa cyclic pressure could up-regulate the expression of osteogenic genes ALP, COL-1, RUNX2, OCN, OPN and OSX in the healthy hPDLSCs. CONCLUSIONS: Collectively, it could be concluded that 0-150 kPa was an excessive stress loading which accelerated both inflammatory and osteoclastic effects, while 0-90 kPa may be a positive factor for the osteogenic differentiation of hPDLCs in vitro.