Potential synergies between matrix proteins and soluble factors on resorption and proteinase activities of rabbit bone cells

Human growth hormone (GH) has recently been found to stimulate osteoclastic resorption, cysteine-proteinase and metalloproteinase activities (MMP-2 and MMP-9) in vitro via insulin-like growth factor-I (IGF-I) produced by stromal cells. The present study investigated the effects of two extracellular matrix components (vitronectin and type-I collagen) on hGH- and hIGF-1-stimulated osteoclastic resorption and proteinase activities in a rabbit bone cell model. After 4 days of rabbit bone cell culture on dentin slices with vitronectin coating, hGH and hIGF-1 stimulated bone resorption and hIGF-1 upmodulated cysteine-proteinase activities. MMP-2 expression (but not resorption, cathepsin or MMP-9 activities) was upmodulated by hGH and hIGF-1 on dentin slices coated with type I collagen as compared to those without coating. Then, vitronectin was synergistic with hIGF-1 in the regulation of cysteine-proteinase production whereas collagen showed synergy with hGH and hIGF-1 in the regulation of MMP-2 production. Anti-alphavbeta3 totally abolished the effects of hGH and hIGF-1 on metalloproteinase release, but had no influence on cathepsin release. The results suggest that cysteine-proteinase modulation is not mediated by alphavbeta3 integrin (strongly expressed on osteoclastic surface) whereas the resorption process and metalloproteinase modulation are clearly mediated by this integrin. Our finding about the collagen coating also suggests that hGH- and hIGF-1-stimulated MMP-2 activity are mediated, along with alphavbeta3 integrin, by another adhesion molecule.     

IL-6, leukemia inhibitory factor,and oncostatin M stimulate bone resorption and regulate the expression of receptor activator of NF-kappa B ligand,osteoprotegerin, and receptor activator of NF-kappa B in mouse calvariae

IL-6, leukemia inhibitory factor (LIF), and oncostatin M (OSM) are IL-6-type cytokines that stimulate osteoclast formation and function. In the present study, the resorptive effects of these agents and their regulation of receptor activator of NF-kappaB ligand (RANKL), RANK, and osteoprotegerin (OPG) were studied in neonatal mouse calvaria. When tested separately, neither human (h) IL-6 nor the human soluble IL-6R (shIL-6R) stimulated bone resorption, but when hIL-6 and the shIL-6R were combined, significant stimulation of both mineral and matrix release from bone explants was noted. Semiquantitative RT-PCR showed that hIL-6 plus shIL-6R enhanced the expression of RANKL and OPG in calvarial bones, but decreased RANK expression. Human LIF, hOSM, and mouse OSM (mOSM) also stimulated 45Ca release and enhanced the mRNA expression of RANKL and OPG in mouse calvaria, but had no effect on the expression of RANK. In agreement with the RT-PCR analyses, ELISA measurements showed that both hIL-6 plus shIL-6R and mOSM increased RANKL and OPG proteins. 1,25-Dihydroxyvitamin D3 (D3) also increased the RANKL protein level, but decreased the protein level of OPG. OPG inhibited 45Ca release stimulated by RANKL, hIL-6 plus shIL-6R, hLIF, hOSM, mOSM, and D3. An Ab neutralizing mouse gp130 inhibited 45Ca release induced by hIL-6 plus shIL-6R. These experiments demonstrated stimulation of calvarial bone resorption and regulation of mRNA and protein expression of RANKL and OPG by D3 and IL-6 family cytokines as well as regulation of RANK expression in preosteoclasts/osteoclasts of mouse calvaria by D3 and hIL-6 plus shIL-6R.     

Modulation by soluble factors of gelatinase activities released by osteoblastic cells

This study investigated the ability of normal human osteoblasts (hOb) and osteogenic sarcoma cells (MG-63 and SaOS2) to produce gelatinases and undergo modulation by interleukin 1beta (IL-1beta), interleukin 6 (IL-6), oncostatin M (OSM), leukaemia inhibitory factor (LIF), growth hormone (GH) and insulin-like growth factor-I (IGF-I). Gelatinase activities were determined by zymogaphy, and a quantitative analysis was performed by ELISA. The MMP-2 activities of the three cell lines were significantly increased in the presence of IL-1beta and IL-6, but no modulation of MMP-2 activities was observed in the presence of OSM, LIF and GH. IGF-I increased the activity released by SaOS2 and hOb, but no modulation was detectable in MG-63 cell conditioned medium. An upmodulation of pro-MMP-2 secretion by SaOS2 and hOb was observed for all soluble factors used, whereas an upmodulation of pro-MMP-2 secretion by MG-63 was observed only in the presence of IL-1beta, IL-6 and IGF-I. Thus, osteoblastic cells modulated by cytokines can be involved in bone resorption as a result of the protease activities released.     

Novel secretion system of recombinant Saccharomyces cerevisiae using an N-terminus residue of human IL-1 beta as secretion enhancer.

An N-terminus sequence of human interleukin 1beta (hIL-1beta) was used as a fusion expression partner for the production of two recombinant therapeutic proteins, human granulocyte-colony stimulating factor (hG-CSF) and human growth hormone (hGH), using Saccharomyces cerevisiae as a host. The expression cassette comprised the leader sequence of killer toxin of Kluyveromyces lactis, the N-terminus 24 amino acids (Ser5-Ala28) of mature hIL-1beta, the KEX2 dibasic endopeptidase cleavage site, and the target protein (hG-CSF or hGH). The gene expression was controlled by the inducible UAS(gal)/MF-alpha1 promoter. With the expression vector above, both recombinant proteins were well secreted into culture medium with high secretion efficiencies, and especially, the recombinant hGH was accumulated up to around 1.3 g/L in the culture broth. This is due presumably to the significant role of fused hIL-1beta as secretion enhancer in the yeast secretory pathway. In our recent report, various immunoblotting analyses have shown that the presence of a core N-glycosylation resident in the hIL-1beta fragment is likely to be of crucial importance in the high-level secretion of hG-CSF from the recombinant S. cerevisiae. When the N-glycosylation was completely blocked with the addition of tunicamycin to the culture, the secretion of hG-CSF and hGH was decreased to a negligible level although the other host-derived proteins were well secreted to the culture broth regardless of the presence of tunicamycin. The N-terminal sequencing of the purified hG-CSF verified that the hIL-1beta fusion peptide was correctly removed by in vivo KEX2 protease upon the exit of fusion protein from Golgi complex. From the results presented in this article, it is strongly suggested that the N-terminus fusion of the hIL-1beta peptide could be utilized as a potent secretion enhancer in the expression systems designed for the secretory production of other heterologous proteins from S. cerevisiae.     

Lysosomal peptidases and glycosidases in rheumatoid arthritis

Lysosomal serine and cysteine proteases are reported to play a role in collagen degradation. In this study, the activities of the lysosomal cysteine proteases cathepsin B and H, dipeptidyl peptidase I, and the serine protease tripeptidyl peptidase I and dipeptidyl peptidase II, all ascribed a role in collagen digestion, were compared with those of the aspartate protease cathepsin D, and lysosomal glycosidases in leukocytes from rheumatoid arthritis patients at different stages of the disease. In all patients the activities of cysteine protease cathepsin B, dipeptidyl peptidase I, aspartate protease cathepsin D, and two glycosidases were elevated, but the activities of the serine proteases tripeptidyl peptidase I, dipeptidyl peptidase II, and the cysteine protease cathepsin H was unchanged. The magnitude of the increased activity was correlated with the duration of the disease. Patients with long-standing RA (10 years or more) had higher cysteine protease activity in their leukocytes than did those with disease of shorter duration. This tendency suggests that elevated lysosomal cysteine protease activities, together with aspartate protease cathepsin D and lysosomal glycosidases (but not serine proteases), are associated with progression of rheumatoid arthritis.     

Potent inhibition of gastric acid secretion by intravenous interleukin-1 beta and -1 alpha in rats.

The influence of human and rat recombinant interleukin-1 (hIL-1 beta and -1 alpha and rIL-1 beta) on acid secretion was investigated in conscious pylorus-ligated rats. Intravenous injection of either hIL-1 beta, hIL-1 alpha or rIL-1 beta dose dependently inhibited gastric acid output with an ED50 of 0.05 microgram, 0.5 microgram and 2.2 micrograms, respectively. The antisecretory action of IL-1 beta was associated with an increase in circulating levels of gastrin. hIL-1 beta-induced inhibition of acid secretion was dose dependently reversed by peripheral injection of the IL-1 receptor antagonist, IL-RA, with a dose ratio of 1:10(3) for complete reversal. The inhibitory effect of hIL-1 beta was blocked by indomethacin and was not modified by IV injections of the CRF receptor antagonist, alpha-helical CRF(9-41), or the monoclonal somatostatin antibody CURE.S6, or by systemic capsaicin pretreatment. These results show that systemic hIL-1 beta-induced inhibition of gastric acid secretion is mediated through IL-1 receptors and prostaglandin pathways, and does not involves CRF receptors, afferent fibers, or changes in circulating gastrin or somatostatin levels.     

Regulation of collagenase activities of human cathepsins by glycosaminoglycans

Cathepsin K, a lysosomal papain-like cysteine protease, forms collagenolytically highly active complexes with chondroitin sulfate and represents the most potent mammalian collagenase. Here we demonstrate that complex formation with glycosaminoglycans (GAGs) is unique for cathepsin K among human papain-like cysteine proteases and that different GAGs compete for the binding to cathepsin K. GAGs predominantly expressed in bone and cartilage, such as chondroitin and keratan sulfates, enhance the collagenolytic activity of cathepsin K, whereas dermatan, heparan sulfate, and heparin selectively inhibit this activity. Moreover, GAGs potently inhibit the collagenase activity of other cysteine proteases such as cathepsins L and S at 37 degrees C. Along this line MMP1-generated collagen fragments in the presence of GAGs are stable against further degradation at 28 degrees C by all cathepsins but cathepsin K, whereas thermal destabilization at 37 degrees C renders the fragments accessible to all cathepsins. These results suggest a novel mechanism for the regulation of matrix protein degradation by GAGs. It further implies that cathepsin K represents the only lysosomal collagenolytic activity under physiologically relevant conditions.     

Action of the Major Protease from Entamoeba histolytica on Proteins of the Extracellular Matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica , a cysteine protease of M, 27,000–29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated α2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Action of the major protease from Entamoeba histolytica on proteins of the extracellular matrix

The action of the major protease from the parasitic protozoon Entamoeba histolytica, a cysteine protease of Mr 27,000-29,000, on some important proteins of the extracellular matrix has been studied. The isolated protease degraded the extracellular matrix proteins from human tissue collagen type IV and V as well as laminin and fibronectin with different velocities and specificities under native conditions. Whereas the degradation of fibronectin and laminin proceeded rapidly, yielding distinct fragment patterns, the breakdown of the collagen types happened more slowly and incompletely. The digestion of the denatured isolated alpha 2-chain of bovine collagen type I was very fast and unspecific requiring only 1/10 of the enzyme activities as compared with the other substrates mentioned above. Nearly 85% of the overall proteolytic activity of a soluble fraction of E. histolytica was strongly inhibited by antibodies against the purified histolytic protease as well as by cystatin from chicken egg white, a specific protein inhibitor of cysteine proteases. We conclude that the histolytic protease represents by far the highest portion of soluble proteolytic activity in E. histolytica which is sufficient to destroy the extracellular matrix of the host.     

Interaction of human breast fibroblasts with collagen I increases secretion of procathepsin B

Interactions of stromal and tumor cells with the extracellular matrix may regulate expression of proteases including the lysosomal proteases cathepsins B and D. In the present study, we determined whether the expression of these two proteases in human breast fibroblasts was modulated by interactions with the extracellular matrix component, collagen I. Breast fibroblasts were isolated from non-malignant breast tissue as well as from tissue surrounding malignant human breast tumors. Growth of these fibroblasts on collagen I gels affected cell morphology, but not the intracellular localization of vesicles staining for cathepsin B or D. Cathepsins B and D levels (mRNA or intracellular protein) were not affected in fibroblasts growing on collagen I gels or plastic, nor was cathepsin D secreted from these cells. In contrast, protein expression and secretion of cathepsin B, primarily procathepsin B, was induced by growth on collagen I gels. The induced secretion appeared to be mediated by integrins binding to collagen I, as inhibitory antibodies against alpha(1), alpha(2), and beta(1) integrin subunits prevented procathepsin B secretion from fibroblasts grown on collagen. In addition, procathepsin B secretion was induced when cells were plated on beta(1) integrin antibodies. To our knowledge, this is the first examination of cathepsin B and D expression and localization in human breast fibroblasts and their regulation by a matrix protein. Secretion of the cysteine protease procathepsin B from breast fibroblasts may have physiological and pathological consequences, as proteases are required for normal development and for lactation of the mammary gland, yet can also initiate and accelerate the progression of breast cancer.     

Regulation of alpha 2(I) collagen expression in stellate cells by retinoic acid and retinoid X receptors through interactions with their cofactors

Retinoic acid (RA) suppresses alpha 2(I) collagen expression in hepatic stellate cells through the binding of retinoic acid receptor beta (RAR beta) and retinoid X receptor alpha (RXR alpha) to RA response elements (RAREs) in the alpha 2(I) collagen promoter. This study determined the influence of coactivators and corepressors to RAR beta and RXR alpha on the regulation of the alpha 2(I) collagen promoter. The coactivators, steroid receptor coactivator-1 (SRC-1) and growth hormone receptor interacting protein-1 (GRIP-1), enhanced, while the nuclear receptor corepressor (N-CoR) abolished the inhibitory effect of RAR beta and RXR alpha on the promoter activity. In the presence of RA, the coactivators SRC-1 and GRIP-1 formed complexes with RAR beta and RXR alpha which are bound to an oligonucleotide specifying a RARE site in the promoter. In conclusion, this study shows that in the presence of retinoic acid, the coactivators SRC-1 and GRIP-1 augment, while the corepressor N-CoR abolishes, the suppressive effects of RAR beta and RXR alpha on alpha 2(I) collagen promoter activity.     

Modulation of fibroblast functions by interleukin 1: increased steady- state accumulation of type I procollagen messenger RNAs and stimulation of other functions but not chemotaxis by human recombinant interleukin 1 alpha and beta

Interleukin-1 (IL-1) is synthesized by and released from macrophages in response to a variety of stimuli and appears to play an essential role in virtually all inflammatory conditions. In tissues of mesenchymal origin (e.g., cartilage, muscle, bone, and soft connective tissue) IL-1 induces changes characteristic of both destructive as well as reparative phenomena. Previous studies with natural IL-1 of varying degrees of purity have suggested that it is capable of modulating a number of biological activities of fibroblasts. We have compared the effects of purified human recombinant (hr) IL-1 alpha and beta on several fibroblast functions. The parameters studied include cell proliferation, chemotaxis, and production of collagen, collagenase, tissue inhibitor of metalloproteinase (TIMP), and prostaglandin (PG) E2. We observed that hrIL-1s stimulate the synthesis and accumulation of type I procollagen chains. Intracellular degradation of collagen is not altered by the hrIL-1s. Both IL-1s were observed to increase the steady-state levels of pro alpha 1(I) and pro alpha 2(I) mRNAs, indicating that they exert control of type I procollagen gene expression at the pretranslational level. We found that both hrIL-1 alpha and beta stimulate synthesis of TIMP, collagenase, PGE2, and growth of fibroblasts in vitro but are not chemotactic for fibroblasts. Although hrIl-1 alpha and beta both are able to stimulate production of PGE2 by fibroblasts, inhibition of prostaglandin synthesis by indomethacin has no measurable effect on the ability of the IL-1s to stimulate cell growth or production of collagen and collagenase. Each of the IL-1s stimulated proliferation and collagen production by fibroblasts to a similar degree, however hrIL-1 beta was found to be less potent than hrIL-1 alpha in stimulating PGE2 production. These observations support the notion that IL-1 alpha and beta may both modulate the degradation of collagen at sites of tissue injury by virtue of their ability to stimulate collagenase and PGE2 production by fibroblasts. Furthermore, IL-1 alpha and beta might also direct reparative functions of fibroblasts by stimulating their proliferation and synthesis of collagen and TIMP.     

Effects of heterologous hematopoietic cytokines on in vitro differentiation of cultured porcine inner cell masses

An exogenous supply of hematopoietic cytokines is essential for maintaining murine embryonic stem (ES) cells in a proliferative yet undifferentiated state. Recently, it was demonstrated that hematopoietic cytokines utilize the gp130 signal transduction pathway to maintain this phenotype, although their involvement toward maintaining porcine ES cell pluripotency has not been established. Therefore, the objective of this study was to determine the effectiveness of several heterologous hematopoietic cytokines at maintaining the isolated porcine inner cell masses (pICM) in an undifferentiated state. pICMs (day 7) were isolated by immunosurgery and cultured 4 days in one of six treatments: control medium, human leukemia inhibitory factor (hLIF; 1,000 u/ml), human interleukin-6 (hlL-6; 100 ng/ml), hlL-6 + hlL-6 soluble receptor (hlL6 + sR; 100 ng/ml + 2.5 μg/ml), human oncostatin M (hOSM; 100 ng/ml), or rat ciliary neurotrophic factor (rCNTF; 100 ng/ml). All cytokines were prepared in Dulbecco's Modified Eagle's Medium/Ham's F-10 (1:1)-based medium. Morphology of plCMs was evaluated on a scale of 1 (fully undifferentiated) to 5 (fully differentiated) at 24-h intervals. Differentiation was significantly lower on day 2 for rCNTF vs. hLIF cultured plCMs (2.07 ± 0.15 vs. 2.70 ± 0.16; P < 0.05). Furthermore, addition of rCNTF gave the lowest overall mean differentiation score (2.53 ± 0.15). However, none of the cytokines significantly delayed differentiation over controls for the 4-day culture period (P > 0.05). Since these heterologous cytokines were unable to inhibit differentiation, it is unlikely they will be beneficial towards isolating porcine ES cell lines under current conditions. Future work with homologous cytokines and dose effects may prove more beneficial. © 1996 Wiley-Liss, Inc.     

Collagenolytic activity of cathepsin K is specifically modulated by cartilage-resident chondroitin sulfates

Cathepsin K is the predominant cysteine protease in osteoclast-mediated bone remodeling, and the protease is thought to be involved in the pathogenesis of diseases with excessive bone and cartilage resorption. Osteoclastic matrix degradation occurs in the extracellular resorption lacuna and upon phagocytosis within the cell's lysosomal-endosomal compartment. Since glycosaminoglycans (GAGs) are abundant in extracellular matrixes of cartilage and growing bone, we have analyzed the effect of GAGs on the activity of bone and cartilage-resident cathepsins K and L and MMP-1. GAGs, in particular chondroitin sulfates, specifically and selectively increased the stability of cathepsin K but had no effect on cathepsin L and MMP-1. GAGs strongly enhanced the stability and, to a lesser extent, the catalytic activity of cathepsin K. To combine the activity and stability parameters, we defined a novel kinetic term, named cumulative activity (CA), which reflects the total substrate turnover during the life span of the enzyme. In the presence of chondroitin-4-sulfate (C-4S), the CA value increased 200-fold for cathepsin K but only 25-fold with chondroitin-6-sulfate (C-6S). C-4S dramatically increased the hydrolysis of soluble as well insoluble type I and II collagens, whereas the effects of C-6S and hyaluronic acid were less pronounced. C-4S acts in a concentration-dependent manner but reaches saturation at approximately 0.1%, a concentration similar to that found in the synovial fluid of arthritis patients. C-4S increased the cathepsin K-mediated release of hydroxyproline from insoluble type I collagen 10-fold but had only a less than 2-fold enhancing effect on the hydrolysis of intact cartilage. The relatively small increase in the hydrolysis of cartilage by C-4S was attributed to the endogenous chondroitin sulfate content present in the cartilage. Although C-4S increased the pH stability at neutral pH, a significant increase in the collagenolytic activity of cathepsin K at this pH was not observed, thus suggesting that the unique collagenolytic activity of cathepsin K at acidic pH is mechanistically determined and not by the enzyme's instability at neutral pH. The selective and significant stabilization and activation of cathepsin K activity by C-4S may provide a rationale for a novel mechanism to regulate the enzyme's activity during bone growth and aging, two processes known for significant changes in the GAG content.     

Regulation of collagenolytic cysteine protease synthesis by estrogen in osteoclasts

In ovariectomized (Ovx) mice, collagenolytic cysteine protease (CCP) activity in calvaria significantly increased 7 days after ovariectomy and was about 50% of that observed in sham-operated (Sham) mice 3 weeks later. In Ovx mice, subcutaneously (s.c.) administered estradiol-17beta (E2) (10 microg/kg) for 2 weeks led to a decrease in CCP activity in calvaria to the level observed in Sham mice. In Ovx mice, though the amount of cathepsin L increased more than that of cathepsin K, cathepsin K and cathepsin L content increased by 200-400% compared with the Sham mice; cathepsin K was detected in larger amounts than cathepsin L in calvaria from both Sham and Ovx mice. The amounts of cathepsin K and cathepsin L in Ovx mice were reduced to the values seen with Sham mice after administration (s.c.) of E2 (10 microg/kg) for 2 weeks. In mouse calvarial organ culture, the increase of CCP activity and release of hydroxyproline, an indicator of degradation of type-I collagen, in the presence of 1alpha,25-(OH)(2)D(3), parathyroid hormone, interleukin (IL)-1alpha, IL-6, or tumor necrosis factor-alpha was suppressed by E2 (10(-9)-10(-7) M). In all cases, secretion of both cathepsin K and cathepsin L were suppressed by E2. In osteoclasts, expression of cathepsin K and cathepsin L was suppressed by E2 at the mRNA level. Cathepsin B was detected faintly or not at all. These results suggest that synthesis of cathepsin K and cathepsin L was negatively regulated by E2 at the mRNA level. In Ovx mice, deficiency of E2 resulted in an augmentation of cathepsin K and cathepsin L synthesis, and the cathepsins might share roles in bone resorption in vivo.     

N-Glycosylation of secretion enhancer peptide as influencing factor for the secretion of target proteins from Saccharomyces cerevisiae

hIL-1beta-derived polypeptide, when fused to the N-terminal end of target proteins, exerts a potent secretion enhancer function in Saccharomyces cerevisiae. We investigated the effect of N-glycosylation of the secretion enhancer peptide on the secretion of target proteins. The N-terminal 24 amino acids (Ser5-Ala28) of human interleukin 1beta (hIL-1beta) and interleukin 1 receptor antagonist (IL-1ra) were used as secretion enhancer for synthesizing recombinant human granulocyte-colony stimulating factor (rhG-CSF) from S. cerevisiae. The mutation of potential N-glycosylation site, by substituting Gln for either Asn7 of N-terminal 24 amino acids of hIL-1beta (Asn7Gln) or Asn84 of IL-1ra (Asn84Gln), resulted in a dramatic reduction of rhG-CSF secretion efficiency. In contrast, the mutant containing an additional N-glycosylation site on the N-terminal 24 amino acids of hIL-1beta (Gln15Asn) secreted twice as much rhG-CSF into culture media as wild type hIL-1beta. These results show that N-glycosylation of the secretion enhancer peptide plays an important role in increasing the secretion efficiency of the downstream target proteins. The results also suggest that judicious choice of enhancer peptide and the control of its glycosylation could be of general utility for secretory production of heterologous proteins from S. cerevisiae.     

Selective inhibition of the collagenase activity of cathepsin K

Cathepsin K, the main bone degrading protease, and chondroitin 4-sulfate (C4-S) form a complex with enhanced collagenase activity. In this report, we demonstrate the specific inhibition of the collagenase activity of cathepsin K by negatively charged polymers without affecting the overall proteolytic activity of the protease. Three different mechanisms to interfere with cathepsin-catalyzed collagen degradation are discussed: 1) inhibition of the formation of the cathepsin K/C4-S complex, 2) inhibition of the attachment of C4-S to collagen, and 3) masking of the collagenase cleavage sites in collagen. By targeting these interaction sites, collagen degradation can be modulated while the non-collagenolytic activities of cathepsin K remain intact. The main inhibitory effect on collagen degradation is due to the impeding effect on the active cathepsin K/C4-S complex. Essential structural elements in the inhibitor molecules are negative charges which compete with the sulfate groups of C4-S in the cathepsin K/C4-S complex. The inhibitory effect can be controlled by length and charge of the polymers. Longer negatively charged polymers (e.g. polyglutamates, oligonucleotides) tend to inhibit all three mechanisms, whereas shorter ones preferentially affect the cathepsin K/C4-S complex.