Masaaki Shima

Regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in osteoblastic cells.

In addition to their stimulating function on osteoclastic bone resorption, bone resorptive factors may regulate proteinases and related factors in osteoblastic cells to degrade bone matrix proteins. This study investigated the regulation of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) by bone resorptive factors in the cultures of mouse osteoblastic MC3T3‐E1 cells, mouse primary osteoblastic (POB) cells, and neonatal mouse calvariae. Expression of either MMP‐2, ‐3, ‐9, ‐11, ‐13, and ‐14 or TIMP‐1, ‐2, and ‐3 was detected in MC3T3‐E1 cells and POB cells. When the bone resorptive factors parathyroid hormone, 1,25‐dihydroxyvitamin D3, prostaglandin E2, interleukin‐1β (IL‐1β), and tumor necrosis factor‐α (TNF‐α) were added to the cell cultures, MMP‐13 mRNA levels were found predominantly to increase by all resorptive factors in the three cultures. mRNA levels of either MMP‐3 and ‐9 or TIMP‐1 and ‐3 were found to increase mainly by the cytokines IL‐1β and TNF‐α. BB94, a nonselective MMP inhibitor, neutralized the 45Ca release stimulated by these resorptive factors to an extent similar to that of calcitonin, strongly suggesting that bone resorptive factors function at least partly through MMP formation. We propose that MMP‐13 mRNA expression in osteoblastic cells may play an important role in stimulating matrix degradation by both systemic and local resorptive factors, whereas either MMP‐3 and ‐9 or TIMP‐1 and ‐3 might modulate matrix degradation by local cytokines only. J. Cell. Physiol. 185:207–214, 2000.

Intrarenal localization of degradation of atrial natriuretic peptide in isolated glomeruli and cortical nephron segments

Using isolated glomeruli and nephron segments obtained from collagenase treated rabbit kidneys, we examined the in vitro degradation of alpha-human atrial natriuretic polypeptide (alpha-hANP). The ANP-degrading activity was measured by the amount of immunoreactive ANP remaining after incubation of about 50 fmoles alpha-hANP with each tissue preparation for 7.5 min. The sequence of degrading activity among isolated nephron segments was as follows: proximal straight tubule greater than proximal convoluted tubule greater than cortical collecting tubule greater than distal convoluted tubule greater than cortical thick ascending limb. A single glomerulus exhibited the degrading activity which was comparable to approximately 50% of the activity of 1 mm proximal convoluted tubule. Phosphoramidon, an inhibitor of endopeptidase, prevented the degradation of ANP in proximal convoluted tubule and glomerulus by 68% and 89%, respectively, but not in cortical thick ascending limb and cortical collecting tubule. From these results, we conclude that the degradation of ANP by endopeptidase occurs mainly in the proximal tubule and glomerulus.

Transcriptional Induction of Matrix Metalloproteinase‐13 (Collagenase‐3) by 1α,25‐Dihydroxyvitamin D3 in Mouse Osteoblastic MC3T3‐E1 Cells

The removal of unmineralized matrix from the bone surface is essential for the initiation of osteoclastic bone resorption because osteoclasts cannot attach to the unmineralized osteoid. Matrix metalloproteinases (MMPs) are known to digest bone matrix. We recently reported that among the MMPs expressed in mouse osteoblastic cells, MMP‐13 (collagenase‐3) was the one most predominantly up‐regulated by bone resorbing factors including 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3]. In this study, we examined the mechanism of regulation of MMP‐13 expression by 1α,25(OH)2D3 in mouse osteoblastic MC3T3‐E1 cells. 1α,25(OH)2D3 increased steady‐state messenger RNA (mRNA) and protein levels of MMP‐13. De novo protein synthesis was essential for the induction because cycloheximide (CHX) decreased the effect of 1α,25(OH)2D3 on the MMP‐13 mRNA level. 1α,25(OH)2D3 did not alter the decay of MMP‐13 mRNA in transcriptionally arrested MC3T3‐E1 cells; however, it increased the MMP‐13 heterogeneous nuclear RNA (hnRNA) level and MMP‐13 transcriptional rate. The binding activity of nuclear extracts to the AP‐1 binding site, but not to the Cbfa1 binding site, in the MMP‐13 promoter region was up‐regulated by 1α,25(OH)2D3, suggesting the mediation of AP‐1 in this transcriptional induction. To determine the contribution of MMPs to bone resorption by 1α,25(OH)2D3, the inhibitory effect of BB94, an MMP inhibitor, on resorbed pit formation by mouse crude osteoclastic cells was examined on either an uncoated or collagen‐coated dentine slice. BB94 did not prevent resorbed pit formation on uncoated dentine whereas it did on collagen‐coated dentine. We therefore propose that the transcriptional induction of MMP‐13 in osteoblastic cells may contribute to the degradation of unmineralized matrix on the bone surface as an early step of bone resorption by 1α,25(OH)2D3.

Novel mutations in the a3 subunit of vacuolar H+-adenosine triphosphatase in a Japanese patient with infantile malignant osteopetrosis

A case of infantile malignant osteopetrosis is described. The patient died from respiratory hemorrhage at 7 months of age despite treatment that included high doses of active vitamin D and administration of interferon-gamma. A postmortem examination revealed the presence of many osteoclasts in the bone, which lacked ruffled borders. This observation was consistent with the histology of bone reported in Atp6i-knockout mice, which lack the gene encoding the a3 subunit of vacuolar-type H(+)-adenosine triphosphatase (ATPase). Sequence analysis of the TCIRG1 gene encoding the a3 subunit revealed two novel mutations: a deletion/insertion mutation in exon 9 and a T-to-C transition at the splice donor site of intron 19. The former mutation caused a frame shift and premature stop codon. The latter was associated with abnormal splicing, which was confirmed by sequencing the products amplified by reverse transcription-polymerase chain reaction (RT-PCR), using total RNA from the liver specimen as template. Although several mutations in the TCIRG1 gene in infantile malignant osteopetrosis have been reported in other populations, this is the first case of a Japanese patient with a mutation identified in this gene. These results support the important role of the subunit in the function of the proton pump.

Hypophosphatemic rickets accompanying McCune-Albright syndrome : evidence that a humoral factor causes hypophosphatemia

Abstract. McCune–Albright syndrome (MAS) is sometimes complicated by hypophosphatemia. However, it remains unclear whether a humoral factor is associated with the cause of hypophosphatemia. We isolated cells with mutations of the Gsα gene from fibrous bone dysplasia tissues of two MAS patients (MAS cells). Severe combined immunodeficiency (SCID) mice were subjected to experiments using from one of these cells patients. Effects of conditioned media (CM) isolated from MAS cells (MAS-CM) on phosphate transport were investigated by using rat renal slices, the renal cell line OK-B, rat intestinal rings and the human intestinal cell line Caco-2. In addition, the effects of MAS-CM on human sodium-dependent phosphate transporter (NPT2) gene promoter activity expression were investigated in the renal cell line OK-B2400 and were compared with the effects of CM isolated from a patient with oncogenic hypophosphatemic osteomalacia (OHO). MAS cells caused significant hypophosphatemia (P < 0.05) and elevated serum alkaline phosphatase activity (P < 0.05) in SCID mice. The MAS-CM significantly inhibited phosphate uptake in everted intestinal rings (P < 0.01), whereas it had no effect on glucose uptake. The MAS-CM had no effect on either phosphate uptake in the kidney or NPT2 gene promoter activity. In contrast, the CM of the OHO patient significantly inhibited phosphate uptake and NPT2 gene promoter activity. These results indicate that the humoral factor derived from fibrous dysplasia cells of the MAS patient is different to that from OHO patients, because the humoral factor from the MAS patient inhibited phosphate transport not in the kidney but in the intestine.

Intranasal absorption of salmon calcitonin

SummaryEight normal subjects and 4 children with osteogenesis imperfecta were administered salmon calcitonin (S-CT) intranasally, and the phamacokinetics of S-CT were studied. In the normal subjects, the plasma S-CT concentration showed a dose-dependent increase over a dosage range of 200–400 IU. Maximal plasma concentrations were reached 20–60 min after intranasal administration of S-CT. The plasma calcium concentration was significantly decreased 60 min after the administration. In the children. S-CT was also absorbed through the nasal mucosa. This suggests that nasal spraying may be an efficient method for administration of S-CT.

Severe mandibuloacral dysplasia caused by novel compound heterozygous ZMPSTE24 mutations in two Japanese siblings

Mandibuloacral dysplasia (MAD) is a rare autosomal recessive progeroid syndrome, characterized by mandibular hypoplasia, acroosteolysis affecting distal phalanges and clavicles, delayed closure of the cranial sutures, atrophic skin, and lipodystrophy. Recently, mutations in lamin A/C (LMNA) and zinc metalloprotease (ZMPSTE24), involved in post‐translational processing of prelamin A to mature lamin A, have been identified in MAD kindreds. We now report novel compound heterozygous mutations in exon 1 (c.121C>T; p.Q41X) and exon 6 (c.743C>T; p.P248L) in ZMPSTE24 in two Japanese sisters, 7‐ and 3‐year old, with severe MAD and characteristic facies and atrophic skin. The older sister had lipodystrophy affecting the chest and thighs but sparing abdomen. Their parents and a brother, who were healthy, had heterozygous mutations. The missense mutation, P248L, was not found in 100 normal subjects of Japanese origin. The mutant Q41X was inactive in a yeast halo assay; however, the mutant P248L retained near normal ZMPSTE24 activity. Immunoblots demonstrated accumulation of prelamin A in the patients’ cell lysates from lymphoblasts. The lymphoblasts from the patients also revealed less intense staining for lamin A/C on immunofluorescence. We conclude that ZMPSTE24 deficiency results in accumulation of farnesylated prelamin A, which may be responsible for cellular toxicity and the MAD phenotype.

Multiple associated endocrine abnormalities in a patient with pseudohypoparathyroidism type 1a

A girl with type 1a pseudohypoparathyroidism (PHP) presented several hormonal abnormalities. Although she had eluded neonatal thyroid screening, she was diagnosed as having hypothyroidism at the age of 5 months. Thereafter, a diagnosis of PHP was made on the basis of skeletal features of Albright osteodystrophy and lack of both cyclic adenosine monophosphate (c-AMP) and phosphaturic responses after parathyroid hormone (PTH) infusion. The basal levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) were higher than normal and showed exaggerated responses to luteinizing hormone-releasing hormone (LH-RH). There was no growth hormone (GH) response to arginine infusion, and the prolactin (PRL) response after thyrotropin-releasing hormone (TRH) infusion was also impaired. The stimulating guanine nucleotide-binding protein (Ns) activity of the erythrocytes was reduced to 66.9%. The skeletal age was not delayed at the age of 5 months in spite of the hypothyroid state, and it advanced following thyroxine and vitamin D treatments.

Cooperation of synthetic insulin-like growth factor I/somatomedin C and 1,25-dihydroxyvitamin D3 on regulation of function in clonal osteoblastic cells.

We investigated the effects of insulin-like growth factor I/somatomedin C (IGF-I/SM-C), and the interaction of IGF-I and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on mouse clonal osteoblasts, MC3T3-E1. IGF-I stimulated [3H]thymidine incorporation into the DNA of the cells at concentrations of 1.3-130 X 10(-9) M. The alkaline phosphatase (ALP) activity in cultures was also raised by the hormone at the same concentrations. The optimal dose of IGF-I was 13 X 10(-9) M. Co-addition of IGF-I (1.3-130 X 10(-9) M) and 1,25(OH)2D3 (10(-11) to 10(-10) M) to the culture of MC3T3-E1 cells caused a synergistic increase in ALP activity. 25(OH)D3 and 24,25(OH)2D3 showed a similar effect with IGF-I at 1000-2000 times higher concentrations than 1,25(OH)2D3. [3H]Proline incorporation into collagenase digestible protein (CDP) in media was stimulated dose-dependently by IGF-I up to 2.2-fold over the control levels at 130 X 10(-9) M. Addition of 1,25(OH)2D3 (5 X 10(-11) M) and IGF-I further elevated the proline incorporation into CDP. However, the increment in CDP synthesis, induced by the two hormones was less than the increment in ALP activity. Thus, we conclude (1) that IGF-I stimulates both cell replication and differentiated functions in cultured murine osteoblasts and (2) that IGF-I and 1,25(OH)2D3 have the synergistic effect on ALP activity and the additive effect on collagen synthesis in MC3T3-E1 cells.

Examination of megalin in renal tubular epithelium from patients with Dent disease

Dent disease is characteristic for the urinary loss of low-molecular-weight proteins and calcium, leading to renal calcification and, in some patients, chronic renal failure. This disorder is caused by loss-of-function mutations in the renal chloride channel gene, CLCN5. The animal model of this disease has demonstrated the possible role of disturbed megalin expression, which is a member of the low-density lipoprotein receptor family and is associated with renal reabsorption of a variety of proteins, in Dent disease. We examined the expression of megalin in the renal tubular epithelium of two unrelated patients with Dent disease. One patient, whose CLCN5 gene was completely deleted, showed significantly decreased staining of megalin compared with controls, while there was no change in another patient with partial deletion of the gene. These results demonstrated that mutation of CLCN5 in some patients with Dent disease may impair the expression of megalin, resulting in abnormal calcium metabolism, manifested as hypercalciuria and nephrocalcinosis.