PERK leads a hub dictating pancreatic ß cell homoeostasis.

In humans, the pathogenesis of diabetes is characterised by two major pancreatic ß cell defects: a reduction in ß cell mass and the failure of ß cells to produce enough insulin. Over the past two decades, multiple studies involving cell cultures, animal models and human subjects have established the importance of the protein kinase RNA-like endoplasmic reticulum kinase (PERK) in the adaptive functional capacity of pancreatic ß cells during embryonic development and into adulthood. In this review, we will highlight major findings identifying PERK as a crucial player in ß cell physiology and in diabetes.

Soluble sulfatase in growing bone of rats.

Soluble sulfatase has been found in epiphyseal, articular, and rib cartilages and in metaphysis and bone marrow of the rat. The greatest activity in young rats is in the metaphysis. Young rats had higher levels of activity in epiphyseal and articular cartilage and in the metaphysis than did the older rats.

Calmodulin-dependent kinase 1beta is expressed in the epiphyseal growth plate and regulates proliferation of mouse calvarial osteoblasts in vitro.

The Ca(2+)/Calmodulin-dependent protein kinase (CaMK) family is activated in response to elevation of intracellular Ca(2+), and includes CaMK1 (as well as CaMK2 and CaMK4), which exists as different isoforms (alpha, beta, gamma and delta). CaMK1 is present in several cell types and may be involved in various cellular processes, but its role in bone is unknown. In situ hybridization was used to determine the spatial and temporal expression of CaMK1beta during endochondral bone development in mouse embryos and newborn pups. The cellular and subcellular distribution of CaMK1 was assessed by quantitative immunogold electron microscopy (EM). The role of CaMK1beta in mouse calvarial osteoblasts was investigated by using small interfering RNA (siRNA) to silence its expression, while in parallel monitoring cell proliferation and levels of skeletogenic transcripts. cRNA in situ hybridization and EM studies show that CaMK1beta is mainly located in developing long bones and vertebrae (from ED14.5 until day 10 after birth), with highest expression in epiphyseal growth plate hypertrophic chondrocytes. By RT-PCR, we show that CaMK1beta2 (but not beta1) is expressed in mouse hind limbs (in vivo) and mouse calvarial osteoblasts (in vitro), and also in primary human articular chondrocyte cultures. Silencing of CaMK1beta in mouse calvarial osteoblasts by siRNA significantly decreases osteoblast proliferation and c-Fos gene expression (approx. 50%), without affecting skeletogenic markers for more differentiated osteoblasts (i.e. Cbfa1/Runx2, Osterix (Osx), Osteocalcin (Oc), Alkaline phosphatase (Alp) and Osteopontin (Opn)). These results identify CaMK1beta as a novel regulator of osteoblast proliferation, via mechanisms that may at least in part involve c-Fos, thus implicating CaMK1beta in the regulation of bone and cartilage development.

Wolcott-Rallison syndrome with 3-hydroxydicarboxylic aciduria and lethal outcome.

Wolcott-Rallison syndrome (WRS) (OMIM 226980) is a rare, autosomal recessive disorder with infancy-onset diabetes mellitus, multiple epiphyseal dysplasia, osteopenia, mental retardation or developmental delay, and hepatic and renal dysfunction as main clinical findings. Patients with WRS have mutations in the EIF2AK3 gene, which encodes the pancreatic eukaryotic translation initiation factor 2-alpha kinase 3. We report a female patient who developed insulin-requiring diabetes at 2.5 months of age. Multiple epiphyseal dysplasia was diagnosed at age 2 years. At age 5.5 years she developed a Reye-like syndrome with hypoketotic hypoglycaemia and renal and hepatic insufficiency and died. A partial autopsy showed fat infiltration in the liver and kidneys. Examination of urine by gas chromatography and mass spectrometry showed large amounts of C(6)-dicarboxylic acid (adipic acid), 3-hydroxy-C(8)-dicarboxylic acid, 3-hydroxy-C(10)-dicarboxylic acid, and 3-hydroxydecenedioic acid. Acetoacetate and 3-hydroxybutyrate were absent. The findings suggested a metabolic block in mitochondrial fatty acid oxidation, but lack of material precluded enzyme analyses. The clinical diagnosis of WRS was suggested in retrospect, and confirmed by sequencing of DNA extracted from stored autopsy material. The patient was compound heterozygous for the novel EIF2AK3 mutations c.1694_1695delAT (Y565X) and c.3044T > C (F1015S). Our data suggest that disruption of the EIF2AK3 gene may lead to defective mitochondrial fatty acid oxidation and hypoglycaemia, thus adding to the heterogeneous phenotype of WRS.

Localization of collagenase in the growth plate of rachitic rats.

In the transition from proliferation to hypertrophic cell zones in the growth plate, there is an increase in chondrocyte volume and a corresponding decrease in collagen content to accommodate the enlarging cells. It is postulated that collagenase accounts for this collagen loss. To test this hypothesis, tibial growth plates were obtained from normal rats, rachitic rats deficient in vitamin D and phosphate, and rats after 48 and 72 h of healing from rickets. Collagenase was quantitated by a pellet assay based on the release of solubilized collagen from the endogenous insoluble collagen in the tissue homogenates. A fourfold greater collagen release and a concomitant sixfold greater hypertrophic cell volume were measured in rachitic growth plates compared with normal age-matched controls. During healing of rickets, collagenase activity and hypertrophic cell volume returned almost to control levels. Rachitic growth plates were dissected into the juxtaepiphyseal 1/3 and the juxtametaphyseal 2/3. The latter portion contained greater than 95% of the hypertrophic cells and 86% of the collagenase. The collagen-degrading activity was extracted from this region and was shown to be a true collagenase by its production of typical A fragments of tropocollagen produced by collagenase action. The enzyme was activated by aminophenylmercuric acetate and trypsin and was inhibited by EDTA, 1,10-phenanthroline, and a tissue inhibitor of metalloproteinases from human articular cartilage. Inhibitors of aspartic, cysteine, and serine proteases had no effect. Micropuncture fluids aspirated from rachitic cartilage contained latent collagenase activity, indicating an extracellular localization. Negative tests for hemoglobin in the rachitic cartilage samples indicated that there was no contamination by capillaries and that this was not a source of collagenase. It is concluded that extracellular collagenase accounts for the loss of cartilage matrix in the hypertrophic zone, and that this process may be distinct from that of capillary invasion.

Protease analysis by neoepitope approach reveals the activation of MMP-9 is achieved proteolytically in a test tissue cartilage model involved in bone formation.

A principle of regulation of matrix metalloproteinase (MMP) activity has been introduced as the cysteine-switch mechanism of activation (Springman et al. 1990). According to this mechanism, a critical Cys residue found in the auto-inhibitory propeptide domain of latent proenzyme is important to determine whether or not activation is turned on or off. The mechanism further allows for multiple modes of activation. To determine whether or not activation is accomplished proteolytically within a rat test cartilage model, protease analysis by the neoepitope approach, which relies upon a set of antibodies, was applied. One is used to identify the MMP-9 proenzyme bearing the critical cysteine residue, the other to identify any enzyme present bearing a new NH2-terminus 89FQTFD. This is indicative of MMP-9 lacking the cysteine switch. The antibody set has been applied to frozen tissue sections and analyzed by light and electron microscopic methods. Results reveal that activation of the MMP-9 protease involves limited proteolysis resulting in propeptide domain release. Here we report the observed changes of protease form to indigenous cells and extracellular matrix, thereby making it possible to uncover the features of MMP-9 activation within a specified set of tissue circumstances where a cartilage model is transformed into definitive bone. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Enzymatic characterization of the chondrocytic alkaline phosphatase isolated from bovine fetal epiphyseal cartilage.

Purified chondrocytic alkaline phosphatase (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) from bovine fetal epiphyseal cartilage hydrolyzes a variety of phosphate esters as well as ATP and inorganic pyrophosphate. Optimal activities for p-nitrophenyl phosphate, ATP and inorganic pyrophosphate are found at pH 10.5, 10.0 and 8.5, respectively. The latter two substrates exhibit substrate inhibition at high concentrations. p-Nitrophenyl phosphate demonstrates decreasing pH optima with decreasng substrate concentration. Heat inactivation studies indicate that both phosphorolytic and pyrophosphorolytic cleavage occur at the same site on the enzyme. Mg2+ (0.1-10.0 mM) and Mn2+ (0.01-0.1 mM) show a small stimulation of p-nitrophenyl phosphate-splitting activity at pH 10.5. Levamisole, Pi, CN-, Zn2+ and L-phenylalanine are all reversible inhibitors of the phosphomonoesterase activity. Pi is a competitive inhibitor with a Ki of 10.0 mM. Levamisole and Zn2+ are potent non-competitive inhibitors with inhibition constants of 0.05 and 0.04 mM, respectively. The chondrocytic alkaline phosphatase is inhibited irreversibly by Be2+, EDTA, EGTA, ethane-1-hydroxydiphosphonate, dichloromethane diphosphonate, L-cysteine, phenyl-methylsulfonyl fluoride, N-ethylmaleimide and iodoacetamide. NaCL, KCL and Na2SO4 at 0.5-1.0 M inhibit the enzyme. At pH 8.5, the cleavage of inorganic pyrophosphate (pyrophosphate phosphohydrolase, EC 3.6.1.1) by the chondrocytic enzyme is slightly enhanced by low levels of Mg2+ and depressed by concentrations higher than 1mM. Ca2+ show only inhibition. Similar effects of Mg2+ and Ca2+ on the associated ATPase (ATP phosphohydrolase, EC 3.1.6.3) activity were observed. Arrhenius studies using p-nitrophenyl phosphate and AMP as substrates have accounted for the ten-fold difference in V in terms of small differences in both the enthalpies and entropies of activation which are 700 cal/mol and 2.3 cal/degree per mol, respectively.

Studies on matrix vesicles isolated from chick epiphyseal cartilage. Association of pyrophosphatase and ATPase activities with alkaline phosphatase.

Fractions composed primarily of cells (Fraction I), membrane fragments (Fraction II) and matrix vesicles (Fraction III) were isolated from chick epiphyseal cartilage. The characteristics of the alkaline phosphatase (EC 3.1.3.1), pyrophosphatase (EC 3.6.1.1) and ATPase (EC 3.6.1.3) activities in the matrix vesicle fraction were studied in detail. Mg-2-+ was not absolutely essential to any of the activities, but at low levels was stimulatory in all cases. Higher concentrations inhibited both pyrophosphatase and ATPase activities. Both the stimulatory and inhibitory effects were pH-dependent. Ca-2-+ stimulated all activities weakly in the absence of Mg-2-+. However, when Mg-2-+ was present, Ca-2-+ was slightly inhibitory. Thus, none of the activities appear to have a requirement for Ca-2-+, and hence would not seem to be involved with active Ca-2-+ transport in the typical manner. The distribution of alkaline phosphatase, pyrophosphatase, and Mg-2-+ ATPase activities among the various cartilage fractions was identical, and concentrated primarily in the matrix vesicles. Conversely, the highest level of (Na-+ + K-+)-ATPase activity was found in the cell fraction. All activites showed nearly identical sensitivities to levamisole (4 - 10-3 M) which caused nearly complete inhibition of alkaline phosphatase and pyrophosphatase. About 10-15% of the ATPase activity was levamisole-insensitive. The data are consistent with the concept that the Mg-2-+-ATPase and pyrophosphatase activities of matrix vesicles stem from one enzyme, namely, alkaline phosphatase.

Isolation and characterization of a metalloprotease associated with chicken epiphyseal cartilage matrix vesicles.

A metalloprotease has been isolated from matrix vesicles of chicken epiphyseal cartilage and subsequently characterized. Matrix vesicles obtained by collagenase digestion and differential centrifugation were further purified by Sepharose CL2B gel filtration. The protease was solubilized from the vesicles by treatment with deoxycholate and freeze-thawing, and then isolated by Sephadex G150 gel filtration. Disc electrophoresis of the enzyme, which displayed protease activity toward azocasein substrate, gave a single protein band. Based on molecular weight (MW) determination, lack of immunocross reactivity, and differences in electrophoretic migration, there is little possibility of any contamination with external protease from the commercial collagenase used for vesicle preparation. The matrix vesicle protease had a MW of 33,000 and a pH optimum of 7.2 and was completely inhibited by 0.1 mM EDTA and 0.2 mM o-phenanthroline. alpha 2-Macroglobulin, ovalbumin, cysteine, penicillamine, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and pyrophosphate at higher concentrations were also inhibitory. The inhibition by omicron-phenanthroline was reversed by Co2+, Zn2+, Fe2+, and Cu2+. Protease activity was most abundant in the heavy fraction of matrix vesicles fractionated by discontinuous sucrose density gradient centrifugation. Release of this protease at the calcifying front could degrade noncollagenous protein moieties that inhibit precipitation of minerals in the extravesicular matrix and thus facilitate mineralization.

Ultrastructural localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) in growth-plate cartilage.

The electron-microscopic cytochemical localization of calcium-activated adenosine triphosphatase (Ca2+-ATPase) was determined in chick epiphyseal growth-plate cartilage. In the reserve zone, mitochondria and lysosomes contained substantial amounts of reaction product, while the plasma membrane and the Golgi complex showed very weak enzymatic activity, and matrix vesicle membranes did not exhibit the cytochemical reaction. As maturation proceeded, the plasma membrane, Golgi complex, and matrix vesicle membranes also stained and were most intense in the proliferative and early hypertrophic zones. From the hypertrophic to the calcifying zone, cytochemical staining decreased progressively in the plasma membrane, the Golgi complex, and lysosomes, while in some cases mitochondrial reaction product remained intense. Matrix vesicles lost their enzymatic activity at the same time that matrix vesicle calcification commenced. It is proposed that this event allows matrix vesicles to calcify, since efflux of calcium would no longer occur.

Anti-idiotypic antibody as an oestrogen mimetic in vivo: stimulation of creatine kinase specific activity in rat animal models.

Previous studies indicated that the anti-idiotypic antibody (clone 1D5) significantly increased the specific activity of creatine kinase (CK) activity in the rat uterus, and in vitro in skeletal cells capable of responding to oestradiol (E2), suggesting that the antibody has oestrogenic-like activity. Moreover, the F(ab')2 dimer of clone 1D5 acted like an antagonist and completely inhibited the increase in CK specific activity by either E2 or clone 1D5 in these skeletal cells. In the present study, we examined the in vivo effects of clone 1D5 and its proteolytic fragment, the F(ab')2 dimer, E2 and dihydrotestosterone (DHT) on CK specific activity in the epiphyseal cartilage, diaphyseal bone, uterus, prostate, thymus and pituitary of immature or gonadectomized female and male rat animal models. In the intact immature animals, clone 1D5 caused an increase in CK in all organs of the female except in the pituitary. In the diaphyseal bone and prostate of male rats there was no stimulation by 1D5. The CK response in the uterus, epiphysis, and diaphysis of immature female rats was dose-dependent and was blocked by either the anti-oestrogen tamoxifen or the F(ab')2 dimer of clone 1D5. E2, DHT, as well as clone 1D5, stimulated CK specific activity in both the diaphysis and epiphysis of ovariectomized female and castrated male rats, whereas sex specificity in the CK response was observed also in the uterus and the prostate of gonadectomized animals. Collectively, these results suggest that, as in cell culture, an intact antibody is necessary for the observed stimulation of CK specific activity and the F(ab')2 dimer can act as an antagonist. Furthermore, the observed biological effects of clone 1D5 which are absolutely parallel to E2, imply that the anti-idiotypic antibody is able to penetrate the cell and reach the nuclear oestrogen receptor and transduces a signal to the nucleus, by as yet uncharacterized mechanisms.

Tissue selective action of tamoxifen methiodide, raloxifene and tamoxifen on creatine kinase B activity in vitro and in vivo.

We have compared the cell and tissue selective estrogenic and antiestrogenic activities of tamoxifen, raloxifene, ICI 164,384 and a permanently ionized derivative of tamoxifen--tamoxifen methiodide (TMI). This non-steroidal antiestrogen has limited ability to cross the blood brain barrier and is therefore less likely to cause the central nervous system disturbances caused by tamoxifen. We have used the stimulation of the specific activity of the "estrogen induced protein", creatine kinase BB, as a response marker in bone, cartilage, uterine and adipose cells and in rat skeletal tissues, uterus and mesometrial adipose tissue. In vitro, TMI, tamoxifen and raloxifene mimicked the agonistic action of 17beta-estradiol in ROS 17/2.8 rat osteogenic osteosarcoma, female calvaria, and SaOS2 human osteoblast cells. In Ishikawa endometrial cancer cells, tamoxifen showed reduced agonistic effects and raloxifene showed no stimulation. However, as antagonists, tamoxifen and raloxifene were equally effective in Ishikawa or SaOS2 cells. In immature rats, all four of the antiestrogens inhibited estrogen action in diaphysis, epiphysis, uterus and mesometrial adipose tissue; when administered alone, tamoxifen stimulated creatine kinase (CK) specific activity in all these tissues. Raloxifene and TMI, however, stimulated only the skeletal tissues and had no stimulatory effect in the uterus or mesometrial fat, and the pure antiestrogen ICI 164,384 showed no stimulatory effect in any of the tissues. The simultaneous injection of estrogen, plus an antiestrogen which acted as an agonist, resulted in lower CK activity than after injection of either agent alone. These differential effects, in vivo and in vitro, may point the way to a wider therapeutic choice of an appropriate antiestrogen which, although antagonizing E2 action in mammary cancer, can still protect against osteoporosis and cardiovascular disease and not stimulate the uterus with its attendant undesirable changes, or interfere with the beneficial action of E2 in the brain.

Changes of femoral alkaline phosphatase activity in adult rats treated by sorbitol enriched or vitamin D3 deficient diet.

The effect of vitamin D3-deficiency and dietary sorbitol on serum calcium level, the activity and alkaline phosphatase (AP) pattern in femoral epiphysis were studied. Rats fed a diet supplemented with sorbitol or vitamin D3 showed the same serum calcium concentration and AP activity in serum and femur. Rats fed a vitamin D3-deficient diet displayed decreased serum calcium concentration and increased AP activity both in serum and femur. Four forms of AP were isolated from the femur of these rat groups: of Mr 100,000, 110,000, 130,000 and 165,000. Rats receiving the diet supplemented with sorbitol showed a marked rise in the activity of the Mr 165,000 form, and appearance of a new monomer of 100,000, never formed in two remaining groups.

Biochemical parameters of osteogenesis in epiphysis of mice irradiated and repopulated with syngeneic bone marrow.

Whole-body irradiation of BALB/cann mice with 600 R of gamma rays produces a profound atrophy of the spleen and diminishes alkaline phosphatase activity in the homogenates of epiphysis. The acid phosphatase activity remains unchanged until day 10, then slightly declines. Incorporation of 45Ca into epiphysis is practically not impaired following irradiation. Repopulation of irradiated animals with 1-1.5 million nucleated syngeneic bone marrow cells restores spleen weight within 6 days, but until day 11 the activity of alkaline phosphatase of epiphysis remains lower. Full recovery of enzyme activities was not observed until day 14, but on day 28 these activities returned to the normal level. Histological inspection did not reveal a full recovery of bone marrow in the epiphysis of irradiated animals until day 14, possibly because of trapping of the vast majority of stem cells by the spleen and liver. On day 28, however, epiphyses were completely filled with the bone marrow.

Immunohistochemical localization of matrix metalloproteinase 13 (MMP-13) in mouse mandibular condylar cartilage.

MMP-13 appears to be one of the most important MMPs in cartilage remodeling and mineralization, because it exhibits a substrate preference for the cartilage-specific type II collagen. The condylar process is constructed by rapid accumulation of hypertrophic chondrocytes during development, but its mechanism is still unclear. To investigate the role of MMP-13 in developing condylar cartilage, we immunohistochemically examined the localization of MMP-13 in the endochondral ossification of the mandibular condyle and tibiae of newborn mice. In the tibiae, the MMP-13 expression was detected only in the deepest layer of the terminal hypertrophic chondrocytes through every examined stage (day 1 to day 10 after birth). On the other hand, in the condylar cartilage at days 1 and 5, MMP-13 was expressed throughout the proliferating and the hypertrophic chondrocytes, and at day 10, MMP-13 was mainly localized in the deepest edge of the hypertrophic layer. A zymographical study showed that the activity of MMP-13 in the condyle was observed at day 1, earlier than in the tibia, and increased until day 7. The time-dependent and cell-specific expression of MMP-13 and its enzymatic property suggest that in the mandibular condylar cartilage, MMP-13 plays a role in making the space for cell enlargement by degradation of the cartilage matrix and in onset of mineralization during the early stage of development.

Role of tartrate-resistant acid phosphatase (TRAP) in long bone development.

Tartrate resistant acid phosphatase (TRAP) was shown to be critical for skeleton development, and TRAP deficiency leads to a reduced resorptive activity during endochondral ossification resulting in an osteopetrotic phenotype and shortened long bones in adult mice. A proper longitudinal growth depends on a timely, well-coordinated vascularization and formation of the secondary ossification center (SOC) of the long bones epiphysis. Our results demonstrate that TRAP is not essential for the formation of the epiphyseal vascular network. Therefore, in wild type (Wt) controls as well as TRAP deficient (TRAP(-/-)) mutants vascularised cartilage canals are present from postnatal day (P) five. However, in the epiphysis of the TRAP(-/-) mice cartilage mineralization, formation of the marrow cavity and the SOC occur prematurely compared with the controls. In the mutant mice the entire growth plate is widened due to an expansion of the hypertrophic zone. This is not seen in younger animals but first detected at week (W) three and during further development. Moreover, an enhanced number of thickened trabeculae, indicative of the osteopetrotic phenotype, are observed in the metaphysis beginning with W three. Epiphyseal excavation was proposed as an important function of TRAP, and we examined whether TRAP deficiency affects this process. We therefore evaluated the marrow cavity volume (MCV) and the epiphyseal volume (EV) and computed the MCV to EV ratio (MCV/EV). We investigated developmental stages until W 12. Our results indicate that both epiphyseal excavation and establishment of the SOC are hardly impaired in the knockouts. Furthermore, no differences in the morphology of the epiphyseal bone trabeculae and remodeling of the articular cartilage layers are noted between Wt and TRAP(-/-) mice. We conclude that in long bones, TRAP is critical for the development of the growth plate and the metaphysis but apparently not for the epiphyseal vascularization, excavation, and establishment of the SOC.

A mathematical model of epiphyseal development: hypothesis of growth pattern of the secondary ossification centre.

This paper introduces a 'hypothesis about the growth pattern of the secondary ossification centre (SOC)', whereby two phases are assumed. First, the formation of cartilage canals as an event essential for the development of the SOC. Second, once the canals are merged in the central zone of the epiphysis, molecular factors are released (primarily Runx2 and MMP9) spreading and causing hypertrophy of adjacent cells. In addition, there are two important molecular factors in the epiphysis: PTHrP and Ihh. The first one inhibits chondrocyte hypertrophy and the second helps the cell proliferation. Between these factors, there is negative feedback, which generates a highly localised and stable pattern over time. From a mathematical point of view, this pattern is similar to the patterns of Turing. The spread of Runx2 hypertrophies the cells from the centre to the periphery of the epiphysis until found with high levels of PTHrP to inhibit hypertrophy. This mechanism produces the epiphyseal bone-plate. Moreover, the hypertrophy is inhibited when the cells sense low shear stress and high pressure levels that maintain the articular cartilage structure. To test this hypothesis, we solve a system of coupled partial differential equations using the finite element method and we have obtained spatio-temporal patterns of the growth process of the SOC. The model is in qualitative agreement with experimental results previously reported by other authors. Thus, we conclude that this model can be used as a methodological basis to present a complete mathematical model of the whole epiphyseal development.