The secretory pathway kinases.

Protein phosphorylation is a nearly universal post-translation modification involved in a plethora of cellular events. Even though phosphorylation of extracellular proteins had been observed, the identity of the kinases that phosphorylate secreted proteins remained a mystery until only recently. Advances in genome sequencing and genetic studies have paved the way for the discovery of a new class of kinases that localize within the endoplasmic reticulum, Golgi apparatus and the extracellular space. These novel kinases phosphorylate proteins and proteoglycans in the secretory pathway and appear to regulate various extracellular processes. Mutations in these kinases cause human disease, thus underscoring the biological importance of phosphorylation within the secretory pathway. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.

Erythrocytic pyruvate kinase mutations causing hemolytic anemia, osteosclerosis, and secondary hemochromatosis in dogs.

BACKGROUND: Erythrocytic pyruvate kinase (PK) deficiency, first documented in Basenjis, is the most common inherited erythroenzymopathy in dogs. OBJECTIVES: To report 3 new breed-specific PK-LR gene mutations and a retrospective survey of PK mutations in as mall and selected group of Beagles and West Highland White Terriers (WHWT). ANIMALS: Labrador Retrievers (2 siblings, 5 unrelated), Pugs (2 siblings, 1 unrelated), Beagles (39 anemic, 29 other),WHWTs (22 anemic, 226 nonanemic), Cairn Terrier (n = 1). METHODS: Exons of the PK-LR gene were sequenced from genomic DNA of young dogs (<2 years) with persistent highly regenerative hemolytic anemia. RESULTS: A nonsense mutation (c.799C>T) resulting in a premature stop codon was identified in anemic Labrador Retriever siblings that had osteosclerosis, high serum ferritin concentrations, and severe hepatic secondary hemochromatosis. Anemic Pug and Beagle revealed 2 different missense mutations (c.848T>C, c.994G>A, respectively) resulting in intolerable amino acid changes to protein structure and enzyme function. Breed-specific mutation tests were developed. Among the biased group of 248 WHWTs, 9% and 35% were homozygous (affected) and heterozygous, respectively, for the previously described mutation (mutant allele frequency 0.26). A PK-deficient Cairn Terrier had the same insertion mutation as the affected WHWTs. Of the selected group of 68 Beagles, 35% were PK-deficient and 3% were carriers (0.37). CONCLUSIONS AND CLINICAL IMPORTANCE: Erythrocytic PK deficiency is caused by different mutations in different dog breeds and causes chronic severe hemolytic anemia, hemosiderosis, and secondary hemochromatosis because of chronic hemolysis and, an as yet unexplained osteosclerosis. The newly developed breed-specific mutation assays simplify the diagnosis of PK deficiency.

A case of hepatitis C-associated osteosclerosis with xanthogranulomatous cholecystitis.

A 62-year-old woman presented with a markedly increased serum ALP level of skeletal origin during a regular follow-up of chronic hepatitis C. Serum calcium, phosphorus, and intact-PTH levels were normal and bone turnover markers were increased. Her generalized bone density was diffusely increased. These findings were consistent with hepatitis C-associated osteosclerosis (HCAO). She underwent cholecystectomy, as gallbladder cancer was suspected; however, histopathological findings demonstrated xanthogranulomatous cholecystitis. After cholecystectomy, serum ALP level and bone turnover markers were gradually decreased. This may indicate the existence of a novel osteogenic factor in the gallbladder in HCAO.

Vitamin D deficiency promotes growth of MCF-7 human breast cancer in a rodent model of osteosclerotic bone metastasis.

INTRODUCTION: Breast cancer metastases to bone are common in advanced stage disease. We have recently demonstrated that vitamin D deficiency enhances breast cancer growth in an osteolytic mouse model of breast cancer metastasis. In this study, we examined the effects of vitamin D deficiency on tumor growth in an osteosclerotic model of intra-skeletal breast cancer in mice. METHODS: The effects of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on proliferation and apoptosis of MCF-7 breast cancer cells, and changes in the expression of genes within the vitamin D metabolic pathway (VDR, 1α- and 24-hydroxylase) were examined in vitro. MCF-7 breast cancer cells were injected intra-tibially into vitamin D deficient and vitamin D sufficient mice co-treated with and without osteoprotegerin (OPG). The development of tumor-related lesions was monitored via serial X-ray analysis. Tumor burden and indices of proliferation and apoptosis were determined by histology along with markers of bone turnover and serum intact PTH levels. RESULTS: In vitro, MCF-7 cells expressed critical genes for vitamin D signalling and metabolism. Treatment with 1,25(OH)(2)D(3) inhibited cell growth and proliferation, and increased apoptosis. In vivo, osteosclerotic lesions developed faster and were larger at endpoint in the tibiae of vitamin D deficient mice compared to vitamin D sufficient mice (1.49±0.08 mm(2) versus 1.68±0.15 mm(2), P<0.05). Tumor area was increased by 55.8% in vitamin D deficient mice (0.81±0.13 mm(2) versus 0.52±0.11 mm(2) in vitamin D sufficient mice). OPG treatment inhibited bone turnover and caused an increase in PTH levels, while tumor burden was reduced by 90.4% in vitamin D sufficient mice and by 92.6% in vitamin D deficient mice. Tumor mitotic activity was increased in the tibiae of vitamin D deficient mice and apoptosis was decreased, consistent with faster growth. CONCLUSION: Vitamin D deficiency enhances both the growth of tumors and the tumor-induced osteosclerotic changes in the tibiae of mice following intratibial implantation of MCF-7 cells. Enhancement of tumor growth appears dependent on increased bone resorption rather than increased bone formation induced by these tumors.

Elevated serum lactate dehydrogenase isoenzymes and aspartate transaminase distinguish Albers-Schönberg disease (Chloride Channel 7 Deficiency Osteopetrosis) among the sclerosing bone disorders.

Osteopetrosis (OPT) refers to the consequences of generalized failure of skeletal resorption during growth. Most cases are explained by loss-of-function mutation within the genes that encode either chloride channel 7 (CLCN7) or a vacuolar proton pump subunit (TCIRG1), each compromising acid secretion by osteoclasts. Patients suffer fractures and sometimes cranial nerve entrapment and insufficient medullary space for hematopoiesis. In 1996, we reported that a high serum level of the brain isoenzyme of creatine kinase (BB-CK), the CK of osteoclasts, characterizes OPT dueamong the sclerosing bone disorders (J Clin Endocrinol Metab. 1996;11:1438). Now, we show that elevation in serum of multiple lactate dehydrogenase (LDH) isoenzymes with aspartate transaminase (AST) distinguishes autosomal dominant OPT due to loss-of-function mutation in CLCN7 [Albers-Schönberg disease (A-SD)] among these conditions. Serum total LDH and AST levels as high as 3× and 2×, respectively, the upper limits of normal for age-appropriate controls, were persistent and essentially concordant in A-SD. Serum LDH was elevated in 7 of 9 children and in the 2 adults studied with A-SD. LDH isoenzyme quantitation showed excesses of LDH-2, -3, and -4. Neither total LDH nor AST increases were found in other forms of OPT, including bisphosphonate-induced OPT, or in 41 children and 6 adults representing 20 additional sclerosing bone disorders. Serum TRACP-5b and BB-CK also were markedly elevated in A-SD. Hence, high serum levels of several enzymes characterize A-SD. Elevated serum LDH isoenzymes and AST indicate a disturbance (of uncertain clinical significance) within multiple extraosseous tissues when there is CLCN7 deficiency.

Cathepsin K inhibitors: a novel target for osteoporosis therapy.

Osteoporosis is characterized by low bone mass with skeletal fragility and an increased risk of fracture. This bone loss is brought about by an imbalance between bone resorption and formation. Cathepsin K is the most abundant cysteine protease expressed in the osteoclast and is believed to be instrumental in bone matrix degradation necessary for bone resorption. Cathepsin K inhibitors represent a novel target for developing agents to treat osteoporosis and other disorders characterized by increased bone resorption.

Angiotensin I-converting enzyme is expressed by erythropoietic cells of normal and myeloproliferative bone marrow.

It is proposed that a locally active, intrinsic renin-angiotensin system (RAS) exists in the bone marrow (BM) and plays a role in regulating haematopoiesis. Angiotensin II type I receptor has been detected on erythroid burst-forming unit-derived cells; its antagonist losartan and angiotensin I-converting enzyme (ACE) inhibitors can suppress erythropoiesis. The possible role of ACE/RAS in BM was investigated by evaluating ACE expression in normal BM, several myeloproliferative disorders and myelodysplasia. Immunohistochemical studies showed that erythroid elements expressed ACE protein in both normal and disturbed haematopoiesis. The presence of ACE in erythroid cells suggests another mechanism for direct ACE inhibitor activity in erythropoiesis.

Lack of vacuolar proton ATPase association with the cytoskeleton in osteoclasts of osteosclerotic (oc/oc) mice.

We examined the pathogenetic mechanism underlying the lack of bone resorption in osteosclerotic oc/oc mice. An immunoelectron microscopic analysis revealed that in the osteoclasts of these mice, no ruffled borders formed, and that vacuolar H+-ATPase (V-ATPase) was present throughout the cytoplasm but not on the apical membranes. The activity of V-ATPase in oc/oc mice was similar to that in normal mice. In normal spleen cell-derived osteoclast-like cells (OCLs), immunoreactivity for V-ATPase was detected in association with Triton X-100-insoluble cellular structure, but not in oc/oc spleen cell-derived OCLs. Moreover, in renal proximal convoluted tubules of oc/oc mice, the basal striation did not form. These results suggest that osteosclerosis in oc/oc mice is possibly due to the dissociation of V-ATPase and cytoskeleton in osteoclasts.

Evidence for an osteoblast-activating factor in a patient with peripheral T-cell lymphoma and osteosclerosis.

A patient with peripheral T-cell Lymphoma and acquired, systemic osteosclerosis is described. Bone histology showed a spectacular activation of osteoblasts accompanyed by massive new bone formation. Alkaline phosphatase in serum was elevated and increased to greater than 2000 U/l when the lymphoma became refractory to chemotherapy. In the patient's serum an osteoblast-activating factor could be demonstrated using a rat osteogenic osteosarcoma cell line (ROS 17/2.8). The factor was absent during remission of the tumor. We conclude that osteosclerosis was a paraneoplastic syndrome in this patient due to the secretion of an osteoblast-stimulating factor by the T-cell lymphoma. This situation is similar to the secretion of osteoclast-activating factors described in B-cell lymphomas, particularly multiple myeloma. The characterization of such a factor could be of therapeutic relevance.