ER Stress During the Pubertal Growth Spurt Results in Impaired Long‐Bone Growth in Chondrocyte‐Specific ERp57 Knockout Mice

Long‐bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57‐dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage‐specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long‐bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan‐rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential. © 2015 American Society for Bone and Mineral Research.     

Targeted Deletion of Autophagy Genes Atg5 or Atg7 in the Chondrocytes Promotes Caspase‐Dependent Cell Death and Leads to Mild Growth Retardation

Longitudinal bone growth takes place in epiphyseal growth plates located in the ends of long bones. The growth plate consists of chondrocytes traversing from the undifferentiated (resting zone) to the terminally differentiated (hypertrophic zone) stage. Autophagy is an intracellular catabolic process of lysosome‐dependent recycling of intracellular organelles and protein complexes. Autophagy is activated during nutritionally depleted or hypoxic conditions in order to facilitate cell survival. Chondrocytes in the middle of the growth plate are hypoxic and nutritionally depleted owing to the avascular nature of the growth plate. Accordingly, autophagy may facilitate their survival. To explore the role of autophagy in chondrocyte survival and constitutional bone growth, we generated mice with cartilage‐specific ablation of either Atg5 (Atg5cKO) or Atg7 (Atg7cKO) by crossing Atg5 or Atg7 floxed mice with cartilage‐specific collagen type 2 promoter–driven Cre. Both Atg5cKO and Atg7cKO mice showed growth retardation associated with enhanced chondrocyte cell death and decreased cell proliferation. Similarly, inhibition of autophagy by Bafilomycin A1 (Baf) or 3‐methyladenine (3MA) promoted cell death in cultured slices of human growth plate tissue. To delineate the underlying mechanisms we employed ex vivo cultures of mouse metatarsal bones and RCJ3.IC5.18 rat chondrogenic cell line. Baf or 3MA impaired metatarsal bone growth associated with processing of caspase‐3 and massive cell death. Similarly, treatment of RCJ3.IC5.18 chondrogenic cells by Baf also showed massive cell death and caspase‐3 cleavage. This was associated with activation of caspase‐9 and cytochrome C release. Altogether, our data suggest that autophagy is important for chondrocyte survival, and inhibition of this process leads to stunted growth and caspase‐dependent death of chondrocytes. © 2015 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).     

Skeletal Mineralization Deficits and Impaired Biogenesis and Function of Chondrocyte‐Derived Matrix Vesicles in Phospho1–/– and Phospho1/Pit1 Double‐Knockout Mice

We have previously shown that ablation of either the Phospho1 or Alpl gene, encoding PHOSPHO1 and tissue‐nonspecific alkaline phosphatase (TNAP) respectively, lead to hyperosteoidosis, but that their chondrocyte‐derived and osteoblast‐derived matrix vesicles (MVs) are able to initiate mineralization. In contrast, the double ablation of Phospho1 and Alpl completely abolish initiation and progression of skeletal mineralization. We argued that MVs initiate mineralization by a dual mechanism: PHOSPHO1‐mediated intravesicular generation of inorganic phosphate (P_i) and phosphate transporter‐mediated influx of P_i. To test this hypothesis, we generated mice with col2a1‐driven Cre‐mediated ablation of Slc20a1, hereafter referred to as P_it1, alone or in combination with a Phospho1 gene deletion. P_it1^col2/col2 mice did not show any major phenotypic abnormalities, whereas severe skeletal deformities were observed in the [Phospho1^–/–; P_it1^col2/col2] double knockout mice that were more pronounced than those observed in the Phospho1^–/– mice. Histological analysis of [Phospho1^–/–; P_it1^col2/col2] bones showed growth plate abnormalities with a shorter hypertrophic chondrocyte zone and extensive hyperosteoidosis. The [Phospho1^–/–; P_it1^col2/col2] skeleton displayed significant decreases in BV/TV%, trabecular number, and bone mineral density, as well as decreased stiffness, decreased strength, and increased postyield deflection compared to Phospho1^–/– mice. Using atomic force microscopy we found that ～80% of [Phospho1^–/–; P_it1^col2/col2] MVs were devoid of mineral in comparison to ～50% for the Phospho1^–/– MVs and ～25% for the WT and P_it1^col2/col2 MVs. We also found a significant decrease in the number of MVs produced by both Phospho1^–/– and [Phospho1^–/–; P_it1^col2/col2] chondrocytes. These data support the involvement of phosphate transporter 1, hereafter referred to as P_iT‐1, in the initiation of skeletal mineralization and provide compelling evidence that PHOSPHO1 function is involved in MV biogenesis. © 2016 American Society for Bone and Mineral Research.     

Thrombomodulin Attenuates Inflammatory Damage Due to Liver Ischemia and Reperfusion Injury in Mice in Toll‐Like Receptor 4–Dependent Manner

Liver ischemia reperfusion injury (IRI) is an important problem in liver transplantation. Thrombomodulin (TM), an effective drug for disseminated intravascular coagulation, is also known to exhibit an anti‐inflammatory effect through binding to the high‐mobility group box 1 protein (HMGB‐1) known as a proinflammatory mediator. We examined the effect of recombinant human TM (rTM) on a partial warm hepatic IRI model in wild‐type (WT) and toll‐like receptor 4 (TLR‐4) KO mice focusing on the HMGB‐1/TLR‐4 axis. As in vitro experiments, peritoneal macrophages were stimulated with recombinant HMGB‐1 protein. The rTM showed a protective effect on liver IRI. The rTM diminished the downstream signals of TLR‐4 and also HMGB‐1 expression in liver cells, as well as release of HMGB‐1 from the liver. Interestingly, neither rTM treatment in vivo nor HMGB‐1 treatment in vitro showed any effect on TLR‐4 KO mice. Parallel in vitro studies have confirmed that rTM interfered with the interaction between HMGB‐1 and TLR‐4. Furthermore, the recombinant N‐terminal lectin‐like domain 1 (D1) subunit of TM (rTMD1) also ameliorated liver IRI to the same extent as whole rTM. Not only rTM but also rTMD1 might be a novel and useful medicine for liver transplantation. This is the first report clarifying that rTM ameliorates inflammation such as IRI in a TLR‐4 pathway–dependent manner.     

Selective CD28 Blockade Attenuates Acute and Chronic Rejection of Murine Cardiac Allografts in a CTLA‐4‐Dependent Manner

Selective blockade of CD28 is a promising therapy to inhibit pathogenic alloimmunity. However, evaluation of this approach in transplantation has been very limited. Using a novel nonactivating single‐chain Fv‐based reagent (α28scFv), we have investigated the role of CD28 and cytotoxic T lymphocyte antigen 4 (CTLA‐4) in a murine cardiac transplant model. Blockade of CD28 for 2 weeks after engraftment promoted allograft survival, and significantly attenuated chronic rejection when combined with transient CD154‐blockade or calcineurin inhibition. Graft acceptance was associated with decreased alloantibody production, increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Blockade of CTLA‐4 during α28scFv‐based treatments led to prompt rejection in all animals and inhibited expression of forkhead box P3 (Foxp3), programmed death (PD)‐1 and 2,3‐indoleamine dioxygenase (IDO) in the graft. These results show that CD28 signaling during the first weeks after transplant is a pivotal mediator of pathogenic alloimmunity, and that selective CD28 blockade prolongs graft acceptance by at least two immunomodulatory mechanisms. Selective CD28 inhibition while sparing CTLA‐4 is thus a promising approach to inhibit pathogenic alloimmunity.