Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1Pcko). S1Pcko mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1Pcko mice. Extraction of type II collagen is substantially lower from S1Pcko cartilage. In S1Pcko mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1Pcko chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

Cartilage is resistant to tumor invasion. In the present study, we found that the NH 2 -propeptide of the cartilage-characteristic collagen, type IIB, PIIBNP, is capable of killing tumor cells. The NH 2 -propeptide is liberated into the extracellular matrix prior to deposition of the collagen fibrils. This peptide adheres to and kills cells from chondrosarcoma and cervical and breast cancer cell lines via the integrins ␣ v ␤ 5 and ␣ v ␤ 3 . Adhesion is abrogated by blocking with anti ␣ v ␤ 5 and ␣ v ␤ 3 antibodies. When ␣ v is suppressed by small intefering RNA, adhesion and cell killing are blocked. Normal chondrocytes from developing cartilage do not express ␣ v ␤ 3 and ␣ v ␤ 5 integrins and are thus protected from cell death. Morphological, DNA, and biochemical evidence indicates that the cell death is not by apoptosis but probably by necrosis. In an assay for invasion, PIIBNP reduced the number of cells crossing the membrane. In vivo, in a tumor model for breast cancer, PIIBNP was consistently able to reduce the size of the tumor.Cartilage is a unique tissue in being composed of only one cell type, chondrocytes. In addition, cartilage is avascular and resistant to tumor invasion, although the mechanism by which this occurs is unknown. Cartilage ECM 2 is predominantly made up of fibrillar type IIB collagen and the large proteoglycan aggrecan. The fibrillar collagens, types I, II, III, V, and XI, are synthesized as procollagens containing NH 2 -and COOH-terminal extension peptides that are removed prior to deposition of the collagen monomers into fibrils in the extracellular matrix (1). During the chondrogenesis phase of endochondral bone development, large amounts of type IIB collagen are synthesized as the tissue is established. Thereafter, in the cartilaginous growth plate, the cells become hypertrophic, change their predominant collagen synthesis to type X collagen, and eventually die by apoptosis. The hypertrophic cartilage is vascularized and subsequently removed by specialized osteoclasts, and the tissue is replaced by bone synthesized by osteoblasts. By this process, the cartilage provides an anlagen for bone formation (2, 3).Type II procollagen is unique among the fibrillar collagens in containing vicinal RGD motifs in the NH 2 -terminal propeptide domain encoded by exon 6 of the COL2A1 gene (supplemental Fig. 1). RGD peptides serve as the primary integrin recognition sites in extracellular matrix proteins and, as such, play an important role in regulating cell/matrix interactions required for proper cell function. Integrins are cell adhesion molecules that consist of two non-covalently associated subunits ␣ and ␤ (4). Integrins are receptors for many ECM matrix proteins, such as for collagens (The binding of substrate to integrins on the cell surface stimulates intracellular signaling to affect gene expression (outside-in signaling), and the cell can alter the expression and affinity of integrins (inside-out signaling). This bidirectional signaling controls cellular activity, such as cell-cell and ce...

Muscle contractile activity results in an increase in glucose uptake rate that can persist for hours. This study was undertaken to determine the effect of carbohydrate repletion on reversal of an exercise-induced increase in glucose uptake. Rats were exercised by swimming. In rats studied 60 min after exercise, muscle glycogen content was 75% depleted and glucose uptake rate was increased. The effect of exercise on glucose uptake was reversed, and glycogen concentration had increased 44 mumol/g muscle, within 18 h in rats fed carbohydrate. In rats fed a carbohydrate-free diet, muscle glycogen increased only 11 mumol/g, and glucose uptake rate had returned only 50% of the way to base line 18 h after exercise. The rate of 3-methylglucose accumulation in muscles was increased sixfold 60 min after exercise. This increase in permeability to sugar was reversed within 18 h in rats fed carbohydrate. In rats fed a carbohydrate-free diet the rate of 3-methylglucose accumulation was still threefold above base line 18 h after exercise. Our results provide evidence that decreased availability of carbohydrate slows reversal of an exercise-induced increase in permeability of muscle to sugar.

Abbreviations used in this paper: Agc1, aggrecan; ATF6, activating transcription factor 6; BiP, immunoglobulin heavy chain binding protein; BSP, bone sialoprotein; Col I, type I collagen; CREBH, cAMP-responsive element binding protein H; E, embryonic day; ERSS, ER stress signaling; IHC, immunohistochemistry; Ihh, Indian hedgehog; MMP, matrix metalloproteinase; OASIS, old astrocyte specifi cally induced substance; PECAM-1, platelet/endothelial cell adhesion molecule 1; Ptc-1, Patched-1; PTHrP, parathyroid hormone-related peptide; S1P, site-1 protease; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element binding protein; WT, wild type.The online version of this article contains supplemental material.

Objective Type IIB procollagen is characteristic of cartilage, comprising 50% of the extracellular matrix. The NH2-propeptide of type IIB collagen, PIIBNP, can kill tumor cells via binding to integrins αVβ3 and αVβ5. As osteoclasts rely on αVβ3 integrins for function in bone erosion, we sought to determine whether PIIBNP could inhibit osteoclast function. Methods We undertook in vitro and in vivo experiments to evaluate both osteoblast and osteoclast function in the presence of recombinant PIIBNP. Adhesion of osteoclasts to PIIBNP was analyzed by staining of attached cells with crystal violet. PIIBNP-induced cell death was evaluated by cell counting Trypan Blue stained cells. The mechanism of cell death was evaluated by DNA fragmentation, TUNEL staining and western blotting to detect cleaved caspases. To determine the role of αVβ3 integrin, osteoclasts were pretreated with αV or β3 integrin specific siRNA before the treatment with PIIBNP. To explore PIIBNP function in vivo, a lipopolysaccharide-induced mouse calvaria lysis model was employed. Results Osteoclasts adhered to PIIBNP via an RGD-mediated mechanism. When osteoclasts were plated on extracellular matrix proteins, PIIBNP induced apoptosis of osteoclasts via caspase 3/8 activation. Osteoblasts and macrophages were not killed. Reduction of αV or β3 integrin levels on osteoclasts by siRNA reduced cell death in a dose-dependent manner. In vivo, PIIBNP could inhibit bone resorption. Conclusion We conclude that PIIBNP can inhibit osteoclast survival and bone resorption via signal transduction through the αVβ3 integrins. Because of this property and the cell specificity, we propose that PIIBNP may play a role in vivo in protecting cartilage from osteoclast invasion and also could be a new therapeutic strategy for decreasing bone loss.