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Post-translational modification of proteins by small ubiquitin-like modifier (SUMO) is known to be involved in a variety of cellular events. This modification, called SUMOylation, is carried out by the E1 activating enzyme, the E2 conjugating enzyme, and multiple E3 ligases. Previous studies have demonstrated that the SUMO E3 ligases, protein inhibitors of activated STAT 1 (PIAS1) and 4 (PIAS4), and the SUMO-targeted ubiquitin ligase, RING finger protein 4 (RNF4), play important roles in the repair of DNA double-strand breaks (DSBs). However, the mechanism by which these SUMO-related enzymes promote DSB repair is still poorly understood. In the present study, we focused on homologous recombination (HR), the most accurate DSB repair pathway, and aimed to elucidate the mechanism by which PIAS1, PIAS4, and RNF4 promote HR. In γ-ray-irradiated normal human fibroblasts, DSB end resection and RAD51 loading, the two essential steps of HR, were significantly impaired by small interfering RNA (siRNA)-mediated depletion of PIAS1, PIAS4, or RNF4. The recruitment of BRCA1, a major HR factor, to DSB sites was reduced in cells depleted of these SUMO-related enzymes. Consistent with the role of BRCA1 in counteracting the p53-binding protein 1 (53BP1)-mediated resection blockade, 53BP1 depletion rescued the reduced resection and RAD51 loading in the cells depleted of PIAS1, PIAS4, or RNF4. Moreover, Rap1-interacting factor 1 (RIF1), a resection inhibitor downstream of 53BP1, became more abundant at DSBs when PIAS1, PIAS4, RNF4, or BRCA1 was depleted. Importantly, the concomitant depletion of BRCA1 with either one of the SUMO-related enzymes did not further increase RIF1 at DSBs, when compared to single depletion of BRCA1. Collectively, these results suggest that PIAS1, PIAS4, RNF4, and BRCA1 work epistatically to counteract 53BP1/RIF1-mediated resection blockade, thereby promoting resection., Competing Interests: Declaration of competing interest None declared., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Oxytocin is known as a social bonding hormone, but it also functions as an anxiolytic or analgesic neurotransmitter. When oxytocin regulates pain or anxiousness centrally as a neurotransmitter, it is secreted by neurons and directly projected to targeted regions. Although the function of oxytocin at the spinal level is well studied, its effects at the supraspinal level are poorly understood. We aimed to investigate the effect of oxytocin at the supraspinal level in vivo using C57BL/6J (wild-type [WT]), oxytocin-deficient (Oxt -/- ), oxytocin receptor-deficient (Oxtr -/- ), and oxytocin receptor-Venus (Oxtr Venus/+ ) mice lines. Response thresholds in Oxtr -/- mice in Hargreaves and von-Frey tests were significantly lower than those in WT mice, whereas open field and light/dark tests showed no significant differences. Moreover, response thresholds in Oxt -/- mice were raised to those in WT mice after oxytocin administration. Following the Hargreaves test, we observed the co-localisation of c-fos with Venus or the oxytocin receptor in the periaqueductal gray (PAG), medial amygdala (MeA), and nucleus accumbens (NAc) regions in Oxtr Venus/+ mice. Furthermore, in the PAG, MeA, and NAc regions, the co-localisation of oxytocin with c-fos and gamma-aminobutyric acid was much stronger in Oxtr -/- mice than in WT mice. However, following von-Frey test, the same findings were observed only in the MeA and NAc regions. Our results suggest that oxytocin exerts its analgesic effect on painful stimulation via the PAG region and a self-protective effect on unpleasant stimulation via the MeA and NAc regions., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Hidehisa Saito reports was provided by Tohoku University Graduate School of Medicine Department of Anaesthesiology and Perioperative medicine, (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Chromosome rearrangements, which are structural chromosomal abnormalities commonly found in human cancer, result from the misrejoining between two or more DNA double-strand breaks arising at different genomic regions. Consequently, chromosome rearrangements can generate fusion genes that promote tumorigenesis. The mechanisms of chromosome rearrangement have been studied using exogenous double-strand break inducers, such as radiation and nucleases. However, the mechanism underlying the occurrence of chromosome rearrangements in the absence of exogenous double-strand break-inducing stimuli is unclear. This study aimed to identify the major source of chromosome rearrangements and the DNA repair pathway that suppresses them. DNA repair factors that potentially suppress gene fusion were screened using The Cancer Genome Atlas dataset. In total, 22 repair factors whose expression levels were negatively correlated with the frequency of gene fusion were identified. More than 60% of these repair factors are involved in homologous recombination, a major double-strand break repair pathway. We hypothesized that DNA single-strand breaks are the source of double-strand breaks that lead to chromosome rearrangements. This study demonstrated that hydrogen peroxide (H 2 O 2 )-induced single-strand breaks gave rise to double-strand breaks in a replication-dependent manner. Additionally, H 2 O 2 induced the formation of RPA and RAD51 foci, which indicated that double-strand breaks derived from single-strand breaks were repaired through homologous recombination. Moreover, treatment with H 2 O 2 promoted the formation of radial chromosomes, a type of chromosome rearrangements, only upon the downregulation of homologous recombination factors, such as BRCA1 and CtIP. Thus, single-strand breaks are the major source of chromosome rearrangements when the expression of homologous recombination factors is downregulated., Competing Interests: Declaration of competing interest None declared., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Combination therapy with glecaprevir and pibrentasvir (PIB) has high efficacy for patients with hepatitis C virus (HCV) infection except among those who experienced NS5A-P32 deletion (del) mutation during prior DAA treatment failure. However, some patients fail to achieve SVR through combination treatment even in the absence of NS5A-P32del. We analyzed emergence of NS5A resistance-associated substitutions (RASs) against PIB using HCV-infected mice. Male human hepatocyte transplanted mice were infected with genotype 1b wild-type HCV. Mice were treated with PIB, resulting in a transient decrease in serum HCV RNA levels but followed by relapse during the treatment. Direct sequence analysis showed emergences of various mutations in the NS5A region, including L31V/P32del, L31F/P32del/Y93H, NS5A-P29del/Y85C, and NS5A-F37Y. PIB was less effective in mice with NS5A-F37Y mutations compared to mice with wild-type HCV. NS5A-F37Y showed 5.4-fold resistance to PIB relative to wild-type based on analysis using HCV subgenomic replicon systems. The present in vivo and in vitro studies identified NS5A-F37Y as a novel RAS against PIB and showed the possibility of emergence of various NS5A RASs including P29del, P32del and F37Y following PIB treatment. These mutations might emerge and lead to failure to respond to DAA therapies including PIB-based regimens in chronic hepatitis C patients., Competing Interests: Declaration of competing interest M.I. has received research funding from Bristol-Myers Squibb and AbbVie. H.A. has received honoraria from Eisai and Bayer. K.C. has received honoraria from Bristol-Myers Squibb and MSD K.K., AbbVie, Gilead Science, Dainippon Sumitomo Pharma and Mitsubishi Tanabe Pharma and research funding from Gilead Science, Dainippon Sumitomo Pharma, MSD K.K., AbbVie, Eisai, TORAY, Otsuka Pharma, Chugai Pharma, Takeda Pharma and Roche., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Chronic postsurgical pain (CPSP) is a serious issue for many postoperative patients. Though there are numerous treatment options for the prevention of CPSP, none of them is optimal as the mechanisms of the transition from acute to chronic postoperative pain have not been elucidated. Ketamine and opioids have been administered for chronic postoperative pain treatment but induce severe adverse reactions and/or physical dependency. Here, we examined whether pre-administration of the nonselective N-methyl-d-aspartate (NMDA) receptor antagonist magnesium sulfate attenuates CPSP behavior and alters the expression of glutamate ionotropic receptor NMDA type subunit 1a (Grin1 mRNA) in a rat skin/muscle incision and retraction (SMIR) model. We assessed the effects of a single subcutaneous magnesium sulfate injection on nociceptive behaviors including guarding pain, mechanical hyperalgesia, and heat hypersensitivity in rats after SMIR surgery. We used reverse transcription-quantitative PCR (RT-qPCR) to evaluate Grin1 mRNA expression in the dorsal horn of the spinal cord on postoperative day 14. Compared with the vehicle, magnesium sulfate administration before SMIR surgery reduced mechanical hyperalgesia for 17 d Grin1 gene expression was significantly higher on the ipsilateral side than the contralateral side (P = 0.001) on postoperative day 14. The magnesium sulfate injection prevented Grin1 mRNA upregulation in the spinal cord dorsal horn. A single magnesium sulfate injection mitigated SMIR-induced mechanical hyperalgesia possibly by modulating Grin1 expression. Preoperative magnesium sulfate administration could prove to be a simple and safe CPSP treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Intraflagellar transport (IFT) is essential for assembling primary cilia required for bone formation. Disruption of IFT frequently leads to bone defects in humans. While it has been well studied about the function of IFT in osteogenic cell proliferation and differentiation, little is known about its role in collagen biosynthesis during bone formation. Here we show that IFT20, the smallest IFT protein in the IFT-B complex, is important for collagen biosynthesis in mice. Deletion of Ift20 in craniofacial osteoblasts displayed bone defects in the face. While collagen protein levels are unaffected by loss of Ift20, collagen cross-linking was significantly altered. In both Ift20:Wnt1-Cre and Ift20:Ocn-Cre mice the bones exhibit increased hydroxylysine-aldehyde deived cross-linking, and decreased lysine-aldehyde derived cross-linking. To obtain insight into the molecular mechanisms, we examined the expression levels of telopeptidyl lysyl hydroxylase 2 (LH2), and associated chaperone complexes. The results demonstrated that, while LH2 levels were unaffected by loss of Ift20, its chaperone, FKBP65, was significantly increased in Ift20:Wnt1-Cre and Ift20:Ocn-Cre mouse calvaria as well as femurs. These results suggest that IFT20 plays a pivotal role in collagen biosynthesis by regulating, in part, telopeptidyl lysine hydroxylation and cross-linking in bone. To the best of our knowledge, this is the first to demonstrate that the IFT components control collagen post-translational modifications. This provides a novel insight into the craniofacial bone defects associated with craniofacial skeletal ciliopathies., Competing Interests: Declaration of competing interest All authors state that they have no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Lysyl hydroxylase 2 (LH2) is an endoplasmic reticulum (ER)-resident enzyme that catalyzes the hydroxylation of lysine residues in the telopeptides of fibrillar collagens. This is a critical modification to determine the fate of collagen cross-linking pathway that contributes to the stability of collagen fibrils. Studies have demonstrated that the aberrant LH2 function causes various diseases including osteogenesis imperfecta, fibrosis, and cancer metastasis. However, surprisingly, a LH2-deficient animal model has not been reported. In the current study, to better understand the function of LH2, we generated LH2 gene knockout mice by CRISPR/Cas9 technology. LH2 deficiency was confirmed by genotyping polymerase chain reaction (PCR), reverse transcriptase-PCR, and immunohistochemical analyses. Homozygous LH2 knockout (LH2 -/- ) embryos failed to develop normally and died at early embryonic stage E10.5 with abnormal common ventricle in a heart, i.e., an insufficient wall, a thin ventricular wall, and loosely packed cells. In the LH2 -/- mice, the ER stress-responsive genes, ATF4 and CHOP were significantly up-regulated leading to increased levels of Bax and cleaved caspase-3. These data indicate that LH2 plays an essential role in cardiac development through an ER stress-mediated apoptosis pathway., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Skin atrophy and delayed wound healing are observed in aged humans; however, the molecular mechanism are still elusive. The aim of this study was to analyze the molecular mechanisms of delayed wound healing by aging using α-Klotho-deficient (kl/kl) mice, which have phenotypes similar to those of aged humans. The kl/kl mice showed delayed wound healing and impaired granulation formation compared with those in wild-type (WT) mice. The skin graft experiments revealed that delayed wound healing depends on humoral factors, but not on kl/kl skin tissue. The mRNA expression levels of cytokines related to acute inflammation including IL-1β, IL-6 and TNF-α were higher in wound lesions of kl/kl mice compared with the levels in WT mice by RT-PCR analysis. LPS-induced TNF-α production model using spleen cells revealed that TNF-α production was significantly increased in the presence of FGF23. Thus, higher levels of FGF23 in kl/kl mouse may have a role to increase TNF-α production in would lesion independently of α-Klotho protein, and impair granulation formation and delay wound healing., (Copyright © 2016 Elsevier Inc. All rights reserved.)

It is well documented that deficiency in ataxia telangiectasia mutated (ATM) protein leads to elevated frequency of chromosome translocation, however, it remains poorly understood how ATM suppresses translocation frequency. In the present study, we addressed the mechanism of ATM-dependent suppression of translocation frequency. To know frequency of translocation events in a whole genome at once, we performed centromere/telomere FISH and scored dicentric chromosomes, because dicentric and translocation occur with equal frequency and by identical mechanism. By centromere/telomere FISH analysis, we confirmed that chemical inhibition or RNAi-mediated knockdown of ATM causes 2 to 2.5-fold increase in dicentric frequency at first mitosis after 2 Gy of gamma-irradiation in G0/G1. The FISH analysis revealed that ATM/p53-dependent G1 checkpoint suppresses dicentric frequency, since RNAi-mediated knockdown of p53 elevated dicentric frequency by 1.5-fold. We found ATM also suppresses dicentric occurrence independently of its checkpoint role, as ATM inhibitor showed additional effect on dicentric frequency in the context of p53 depletion and Chk1/2 inactivation. Epistasis analysis using chemical inhibitors revealed that ATM kinase functions in the same pathway that requires kinase activity of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) to suppress dicentric frequency. From the results in the present study, we conclude that ATM minimizes translocation frequency through its commitment to G1 checkpoint and DNA double-strand break repair pathway that requires kinase activity of DNA-PKcs., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The cohesin loading factor NIPBL is required for cohesin to associate with chromosomes and plays a role in DNA double-strand break (DSB) repair. Although the NIPBL homolog Scc2 is recruited to an enzymatically generated DSB and promotes cohesin-dependent DSB repair in yeast, the mechanism of the recruitment remains poorly understood. Here we show that the human NIPBL is recruited to the sites of DNA damage generated by micro-irradiation as well as to the sites of DSBs induced by homing endonuclease, I-PpoI. The recruitment of NIPBL was impaired by RNAi-mediated knockdown of MDC1 or RNF168, both of which also accumulate at DSBs. We also show that the recruitment of NIPBL to the sites of DNA damage is mediated by its C-terminal region containing HEAT repeats and Heterochromatin protein 1 (HP1) interacting motif. Furthermore, NIPBL accumulation at damaged sites was also compromised by HP1γ depletion. Taken together, our study reveals that human NIPBL is a novel protein recruited to DSB sites, and the recruitment is controlled by MDC1, RNF168 and HP1γ., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Recently, significant attention has been drawn to the biology of small leucine-rich repeat proteoglycans (SLRPs) due to their multiple functionalities in various cell types and tissues. Here, we characterize a novel SLRP member, "Podocan-like (Podnl) protein" identified by a bioinformatics approach. The Podnl protein has a signal peptide, a unique cysteine-rich N-terminal cluster, 21 leucine-rich repeat (LRR) motifs, and one putative N-glycosylation site. This protein is structurally similar to podocan in SLRPs. The gene was highly expressed in mineralized tissues and in osteoblastic cells and the high expression level was observed at and after matrix mineralization in vitro. Podnl was enriched in newly formed bones based on immunohistochemical analysis. When Podnl was transfected into osteoblastic cells, the protein with N-glycosylation was detected mainly in the cultured medium, indicating that Podnl is a secreted N-glycosylated protein. The endogenous Podnl protein was also present in bone matrix. These data provide a new insight into our understanding of the emerging SLRP functions in bone formation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

To provide further insights into non-genomic action of thyroid hormone (T3), we investigated whether Src is under control of T3 in primary calvarial osteoblasts prepared from neonatal mice. Treatment of the cells with T3 rapidly decreased Src Y416 autophosphorylation, followed by the decrease of phosphorylated extracellular signal-regulated kinases, suggesting that T3 non-genomically suppresses Src activity. Furthermore, this T3 effect was rapid and persistent, and was associated with the increased expression of osteocalcin (OC). To confirm the contribution of Src to the effect of T3 on OC expression, a constitutively active Src (Y527F) was overexpressed in osteoblasts. In such cells, Y416 phosphorylation was markedly increased even in the presence of T3, and T3-dependent expression of OC was markedly attenuated. The present study demonstrates a novel, non-genomic action of T3 in primary mouse osteoblasts, by which T3 suppresses Src thereby stimulating OC expression.

We used osteoblastic MC3T3-E1 cells to clarify the mechanisms by which dexamethasone (Dex) suppresses osteoblast function, or alendronate or parathyroid hormone (PTH) alleviate it. Dex (10(-7)M) increased mRNA expression of bone morphogenetic protein (BMP) antagonists, follistatin and Dan, and of a Wnt antagonist, secreted frizzled-related protein-1 (sFRP-1) and a Wnt signal inhibitor, axin-2, while concomitantly decreased the expression of downstream molecules, Runx2 mRNA and beta-catenin protein. Pretreatments with alendronate (10(-8)M) or human PTH-(1-34) (10(-8)M) totally or partially antagonized not only the Dex-induced enhancement in mRNA expression of follistatin/Dan and sFRP-1/axin-2 but also the Dex-induced reduction in Runx2 mRNA expression and mineralization. These findings suggest that Dex suppresses the Wnt and BMP pathways as well as osteoblast function by enhancing the expression of BMP and Wnt antagonists, and bisphosphonate and PTH exert pharmacologic effects by canceling these processes.

The active form of vitamin D, 1,25(OH)(2)D(3), has a broad range of effects on bone, however, its role in the quality of bone matrix is not well understood. In this study, using an osteoblastic cell (MC3T3-E1) culture system, the effects of 1,25(OH)(2)D(3) on collagen cross-linking and related enzymes, i.e., lysyl hydroxylases (LH1-3) and lysyl oxidases (LOX, LOXL1-4), were examined and compared to controls where cells were treated with cholecalciferol or ethanol. When compared to the controls, gene expressions of LH1, LH2b and LOXL2 were significantly upregulated by 1,25(OH)(2)D(3) up to 72h of culture. In addition, hydroxylysine (Hyl), Hyl aldehyde (Hyl(ald)), Hyl(ald)-derived cross-links and a total number of cross-links of collagen were significantly higher and the cross-link maturation was accelerated in the 1,25(OH)(2)D(3) treated group. These results demonstrate that 1,25(OH)(2)D(3) directly regulates collagen cross-linking in this culture system likely by upregulating gene expression of specific LH and LOX enzymes.

It is unclear whether metformin, one of the anti-hyperglycemic agents commonly used for type 2 diabetes, could affect bone formation through activation of AMP-activated protein kinase (AMPK). In order to clarify this issue, we investigated the effects of metformin on the differentiation and mineralization of osteoblastic MC3T3-E1 cells as well as intracellular signal transduction. Metformin (50 microM) significantly increased collagen-I and osteocalcin mRNA expression, stimulated alkaline phosphatase activity, and enhanced cell mineralization. Moreover, metformin significantly activated AMPK in dose- and time-dependent manners, and induced endothelial nitric oxide synthase (eNOS) and bone morphogenetic protein-2 (BMP-2) expressions. Supplementation of Ara-A (0.1mM), a specific AMPK inhibitor, significantly reversed the metformin-induced eNOS and BMP-2 expressions. Our findings suggest that metformin can induce the differentiation and mineralization of osteoblasts via activation of the AMPK signaling pathway, and that this drug might be beneficial for not only diabetes but also osteoporosis by promoting bone formation.

The pattern of collagen cross-linking is tissue specific primarily determined by the extent of hydroxylation and oxidation of specific lysine residues in the molecule. In this study, murine pre-myoblast cell line, C2C12 cells, were transdifferentiated into osteoblastic cells by bone morphogenetic protein (BMP)-2 treatment, and the gene expression of lysyl hydroxylases (LH1, 2a/b, and 3) and lysyl oxidase (LOX)/lysyl oxidase-like proteins (LOXL1-4), and the extent of hydroxylysine were analyzed. After 24h of treatment, the expression of most isoforms were upregulated up to 96h whereas LH2a and LOXL2 decreased with time. In the treated cells, both hydroxyproline and hydroxylysine were detected at day 7 and increased at day 14. The ratio of hydroxylysine to hydroxyproline was significantly increased at day 14. The results indicate that LHs and LOX/LOXLs are differentially responsive to BMP-induced osteoblast differentiation that may eventually lead to the specific collagen cross-linking pattern seen in bone.

We report here that ectopically expressed wild-type p53 protein showed more than 6 times longer half-life than normal human fibroblasts in NCl-H1299, a widely used cell line derived from non-small cell lung carcinoma lacking the expression of p53 protein. We found no abnormality in the phosphorylation and ubiquitination of p53, and the expression levels of MDM2. Although proteasome activity measured in vitro was not significantly different between the tumor cell line and normal human fibroblasts, proteasome inhibitors, ALLN, MG115, and MG132, did not accumulate p53 protein in the tumor cell line, but did accumulate p53 in normal human cells. These results provide a novel mechanism, by which p53 is stabilized in tumor cells, and they suggest that a mediator should exist between ubiquitinated p53 and proteasome, which may be defective in H1299 cells.

Covalent intermolecular cross-linking of collagen is initiated by the action of lysyl oxidase (LOX) on the telopeptidyl lysine and hydroxylysine residues. Recently, several LOX isoforms, i.e., LOX-like proteins 1-4 (LOXL1-4), have been identified but their specific tissue distribution and functions are still largely unknown. In this study, mRNA expression of LOX and LOXL1-4 in MC3T3-E1 osteoblastic cells was screened by RT-PCR and quantitatively analyzed by real-time PCR during cell differentiation and matrix mineralization. The results demonstrated that LOX and all LOXLs, except LOXL2, were expressed in this cell line and that the expression pattern during cell differentiation and matrix mineralization was distinct from one another. This indicates that the expression of LOX and its isoforms is highly regulated during osteoblast differentiation, suggesting their distinct roles in collagen matrix stabilization and subsequent mineralization.

Phosphorylation of p53 at Ser15, Thr18, and Ser20 has been thought to be important for p53 stabilization in response to ionizing radiation. In the present study, we examined the X-ray-induced stabilization of Ala-substituted p53 protein at Ser15, Thr18, and Ser20, whose gene expression was controlled under an ecdyson-inducible promoter. We found that all single-, double-, or triple-Ala-substituted p53 at Ser15, Yhr18, and Ser20 were accumulated in the nucleus similarly to wild-type p53 after X-irradiation. These results indicate that the phosphorylation of p53 at Ser15, Thr18, and Ser20 is not necessarily needed for p53 stabilization in response to ionizing radiation.

Regulation of the bone sialoprotein (BSP) gene is important in the differentiation of osteoblasts, in bone matrix mineralization, and in tumor metastasis. We investigated BSP gene transcription by performing functional analysis of the 9256bp of the 5' flanking region of the murine BSP gene containing its promoter. We found that the forced expression of BSP stimulated mouse BSP promoter activity in a dose-dependent manner in both MC3T3-E1 preosteoblast and HEK-293 cell lines, which was transcriptional factor Cbfa1 independent. Co-culture of cells separately expressing BSP promoter reporter and BSP failed to mediate the BSP autoregulation, suggesting that the event might happen intracellularly. Deletion analysis of the BSP promoter indicated that the proximal promoter (110bp) was sufficient to confer this autoregulation. We conclude that the BSP gene is autoregulated in part by a positive feedback on its own promoter.

The lysine (Lys) hydroxylation pattern of type I collagen produced by human fibroblasts in culture was analyzed and compared. Fibroblasts were cultured from normal human skin (NSF), keloid (KDF), fetal skin (FDF), and skin tissues of Ehlers-Danlos syndrome type VIA and VIB patients (EDS-VIA and -VIB). The type I collagen alpha chains with or without non-helical telopeptides were purified from the insoluble matrix and analyzed. In comparison with NSFs, KDF and FDF showed significantly higher Lys hydroxylation, particularly in the telopeptide domains of both alpha chains. Both EDS-VIA and -VIB showed markedly lower Lys hydroxylation in the helical domains of both alpha chains whereas that in the telopeptides was comparable with those of NSFs. A similar profile was observed in the tissue sample of the EDS-VIB patient. These results demonstrate that the Lys hydroxylation pattern is domain-specific within the collagen molecule and that this method is useful to characterize the cell phenotypes in normal/pathological connective tissues.

Decorin (DCN), a member of small leucine-rich proteoglycans, is known to modulate collagen fibrillogenesis. In order to investigate the potential roles of DCN in collagen matrix mineralization, several stable osteoblastic cell clones expressing higher (sense-DCN, S-DCN) and lower (antisense-DCN, As-DCN) levels of DCN were generated and the mineralized nodules formed by these clones were characterized. In comparison with control cells, the onset of mineralization by S-DCN clones was significantly delayed; whereas it was markedly accelerated and the number of mineralized nodules was significantly increased in As-DCN clones. The timing of mineralization was inversely correlated with the level of DCN synthesis. In these clones, the patterns of cell proliferation and differentiation appeared unaffected. These results suggest that DCN may act as an inhibitor of collagen matrix mineralization, thus modulating the timing of matrix mineralization.

The calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays key roles in extracellular calcium ion (Ca(2+)(o)) homeostasis by enabling parathyroid, kidney, and other cells to directly "sense" changes in Ca(2+)(o). In multiple myeloma-associated bone disease, myeloma cells could raise the level of Ca(2+)(o) within their immediate vicinity in the bone marrow microenvironment, through their known capacity to cause bone destruction by stimulating osteoclastic bone resorption. Thus if myeloma cells expressed the CaR, they might sense these locally elevated levels of Ca(2+)(o), which could, in turn, potentially modify their function(s) in ways that could contribute to myeloma bone disease or other aspects of the pathophysiology of this disabling hematological malignancy. In this study, we examined the expression of the CaR in three myeloma cell lines, human U266, IM-9, and RPMI8226 cells. CaR protein was present in all three cell lines as assessed by immunocytochemistry and Western blot analysis using a monoclonal antibody specific for the CaR. Moreover, the use of reverse transcription-polymerase chain reaction (RT-PCR) with CaR-specific primers, followed by nucleotide sequencing of the amplified products, also identified CaR transcripts in the three cell lines. Exposure to known polycationic agonists of the CaR, including high Ca(2+)(o) (2.5mM), neomycin, and gadolinium (Gd(3+)) as well as a specific CaR activator, NPS R467, augmented cell proliferation in all three cell lines. RT-PCR revealed that U266 cells, but not IM-9 cells or RPMI8226 cells, expressed interleukin-6 (IL-6), the expression of which was not enhanced by treatments with CaR agonists. Therefore, taken together, our data first document the fact that the myeloma cell lines, U266, IM-9, and RPMI8226, all express CaR protein and mRNA. Moreover, the CaR expressed on myeloma cells could sense the locally high levels of Ca(2+)(o) in the vicinity of sites of osteoclastic bone resorption and stimulate their proliferation in an IL-6-independent manner. These processes may result in promoting further growth of the tumor and aggravating the associated bone disease.

A novel small gene, designated hepatocyte growth factor activator inhibitor type 2 (HAI-2)-related small peptide (H2RSP) was cloned and characterized in the process of the search for splicing variant forms of HAI-2 by 3'-rapid amplification of cDNA ends (RACE). The gene consisted of 4 exons spanning approximately 1 kb and was located in 11 kb downstream of HAI-2 gene (19q.13.11). The novel transcript identified by 3'-RACE was thought to be chimerically transcribed from both HAI-2 (exons 1-7) and H2RSP (exons 2-4) genes. Wild-type H2RSP mRNA (0.5 kb) was detected abundantly in various tissues including the gastrointestinal tract, whereas chimeric mRNA (1.5 kb) was found mainly in the kidney, prostate, and placenta by Northern blot analysis. The predicted amino acid sequence of H2RSP contained two unique domains, namely the serine-rich region (exon 3) and the lysine-rich region (exon 4). Transfection of deleted series of H2RSP cDNAs fused to enhanced green fluorescent protein (EGFP) into HeLa cells revealed that H2RSP has nuclear localization signal in the lysine-rich region. Immunohistochemical study using anti-H2RSP polyclonal antibody indeed revealed the nuclear localization of this peptide in vivo. These results suggest that H2RSP and H2RSP/HAI-2 chimeric peptides might function as a transcriptional regulatory peptide at the nucleus., (Copyright 2001 Academic Press.)

Keloid is a tissue with an excessive accumulation of collagen. In this study, we have partially characterized post-translational modifications of type I collagen in human keloid in order to pursue their potential involvement in this pathology. The levels of lysyl hydroxylation of the helical portions of alpha 1 and alpha 2 chains of type I collagen in keloid were significantly higher than those of normal, while the levels of prolyl hydroxylation were identical between these two groups. The contents of the major reducible cross-links in dermal collagen, dehydro-hydroxylysinonorleucine and dehydro-histidinohydroxymero-desmosine, were both significantly higher in keloids (up to sixfold) than those of normal. In addition, significant amounts of hydroxylysine-aldehyde derived cross-links that are characteristic of skeletal tissue collagens, dehydro-dihydroxylysinonorleucine (about 0.3 mole/mole of collagen) and pyridinoline (about 0.1 mole/mole of collagen), were found in keloids. These results indicate that keloid-forming cells are phenotypically different from those in normal dermis and that the collagen produced is highly cross-linked. The increased cross-linking provides the fibrils with more stability that may result in an accumulation of collagen.

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder that is characterized by the appearance of multiple outgrowths of the long bones (exostoses) at their epiphyses. Genetical heterogeneities have segregated at least on chromosome 8, 11, and 19 and been designated EXT1, EXT2, and EXT3, respectively. Recently, the responsible genes for EXT1 and EXT2 have been isolated and appeared to define a structurally related gene family. In the present study, we have identified novel genes which share significant sequence homologies with the EXT genes. The predicted protein products of the novel EXT-related genes, EXTR and EXTR2 (for EXT-related genes 1 and 2), consist of 919 and 330 amino acid residues, respectively. These genes were transcribed ubiquitously in various tissues. Based on PCR-assisted analyses of both a human/rodent mono-chromosomal hybrid cell panel and a radiation hybrid mapping panel, EXTR1 was localized to the chromosome 8p21 region, where loss of heterozygosity has been frequently observed in various tumors, and EXTR2 was assigned to the chromosome 1p21 region, where osteopetrosis, a dominant hereditary disease of bone, has been mapped by genetic linkage analysis, implying that the protein products of these two EXT-related genes, as well as of the EXT genes, have potential tumor suppressor activity.

Collagen tryptic peptides obtained from the nonmineralized and mineralized compartments of diaphyseal bone have different distributions of intermolecular crosslinks. Pyridinoline, a collagen crosslink thought to be associated with chronologically older bone, was detected in peptides from nonmineralized collagen but not from mineralized collagen. Mineralization may prevent collagen maturation; conversely, pyridinoline in nonmineralized collagen may decrease the intermolecular distances among collagen chains in fibrils and preclude mineralization.

This report represents a clear demonstration of a cross-link in collagen whose abundance is related to chronological aging of an organism. Recently its structure was identified as histidinohydroxylysinonorleucine. Quantification of the cross-link in various aged samples of bovine and human skin indicate that it rapidly increases from birth through maturation. Subsequently, a steady increase occurs with aging, approaching 1 mole/mole of collagen. This compound seems to be related to the relative proportions of soluble to insoluble collagen from skin in neutral salt, dilute acid, and denaturing aqueous solvents (higher concentration in the insoluble portion). It is absent from other major collagenous tissues such as dentin, bone and tendon.