Human Umbilical Cord Mesenchymal Stem Cell-Derived Extracellular Vesicles Carrying MicroRNA-29a Improves Ovarian Function of Mice with Primary Ovarian Insufficiency by Targeting HMG-Box Transcription Factor/Wnt/ß-Catenin Signaling.

Background: Primary ovarian insufficiency (POI) is a female disease characterized by ovarian function loss under 40 years old. Transplantation of exosomes is an encouraging regenerative medicine method that has the potential for restoring ovarian functions post-POI with high efficiency. Therefore, we investigate the therapeutic efficacy and potential mechanisms of human umbilical cord mesenchymal stem cell- (UCMSC-) derived exosomes on ovarian dysfunction post-POI. Methods: The model of POI was established by intraperitoneal injection with 5 mg/kg cisplatin. The mouse ovarian function was detected by measuring the levels of anti-Mullerian hormone, follicle-stimulating hormone, and estradiol and detecting the morphological changes. For in vitro experiments, the characterization and identification of UCMSCs and UCMSC-derived exosomes were done by observation of morphologies and flow cytometry. To exclude the interference effect of nonspecific precipitation substances, UCMSCs were treated with RNase A or RNase A in combination with Triton X-100. Granulosa cell (GC) identification was performed using immunofluorescence. GC proliferation and viability were assessed using 5-ethynyl-2'-deoxyuridine (EdU) assays and Cell Counting Kit-8 (CCK-8), and GC apoptosis was calculated by flow cytometry. Gene expression and protein levels were evaluated using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting. The binding relationship between miR-29a and HMG-box transcription factor (HBP1) was verified by luciferase reporter assays. Results: In vitro, the human UCMSC-derived exosomes carrying miR-29a upregulation promoted the proliferation of GCs and suppressed their apoptosis. In vivo, miR-29a upregulation reserved the mature follicles and restored the ovarian functions. miR-29a targeted HBP1 and negatively regulated its expression. HBP1 upregulation rescued the miR-29a upregulation-induced inhibition in GC apoptosis and inactivated the Wnt/ß-catenin pathway. Conclusion: The exosomal miR-29a derived from human UCMSCs improves the ovarian function by targeting HBP1 and activating the Wnt/ß-catenin pathway.

HMGB1 in renal ischemic injury.

Factors that initiate cellular damage and trigger the inflammatory response cascade and renal injury are not completely understood after renal ischemia-reperfusion injury (IRI). High-mobility group box-1 protein (HMGB1) is a damage-associated molecular pattern molecule that binds to chromatin, but upon signaling undergoes nuclear-cytoplasmic translocation and release from cells. Immunohistochemical and Western blot analysis identified HMGB1 nuclear-cytoplasmic translocation and release from renal cells (particularly vascular and tubular cells) into the venous circulation after IRI. Time course analysis indicated HMGB1 release into the venous circulation progressively increased parallel to increased renal ischemic duration. Ethyl pyruvate (EP) treatment blocked H(2)O(2) (oxidative stress)-induced HMGB1 release from human umbilical vein endothelial cells in vitro, and in vivo resulted in nuclear retention and significant blunting of HMGB1 release into the circulation after IRI. EP treatment before IRI improved short-term serum creatinine and albuminuria, proinflammatory cyto-/chemokine release, and long-term albuminuria and fibrosis. The renoprotective effect of EP was abolished when exogenous HMGB1 was injected, suggesting EP's therapeutic efficacy is mediated by blocking HMGB1 translocation and release. To determine the independent effects of circulating HMGB1 after injury, exogenous HMGB1 was administered to healthy animals at pathophysiological dose. HMGB1 administration induced a rapid surge in systemic circulating cyto-/chemokines (including TNF-α, eotaxin, G-CSF, IFN-γ, IL-10, IL-1α, IL-6, IP-10, and KC) and led to mobilization of bone marrow CD34+Flk1+ cells into the circulation. Our results indicate that increased ischemic duration causes progressively enhanced HMGB1 release into the circulation triggering damage/repair signaling, an effect inhibited by EP because of its ability to block HMGB1 nuclear-cytoplasmic translocation.

High-mobility group protein HMGA2-derived fragments stimulate the proliferation of chondrocytes and adipose tissue-derived stem cells.

In previous research, it was shown that recombinant HMGA2 protein enhances the proliferation of porcine chondrocytes grown in vitro, opening up promising applications of this embryonic architectural transcription factor for tissue engineering, such as in cartilage repair. In this paper, we describe the development and analyses of two synthetic fragments comprising the functional AT-hook motifs of the HMGA2 protein, as well as the nuclear transport domain. They can be synthesised up to large scales, while eliminating some of the problems of recombinant protein production, including unwanted modification or contamination by the expression hosts, or of gene therapy approaches such as uncontrolled viral integration and transgene expression even after therapy. Application of one of these peptides onto porcine hyaline cartilage chondrocytes, grown in in vitro monolayer cell culture, showed a growth-promoting effect similar to that of the wild type HMGA2 protein. Furthermore, it also promoted cell growth of adult adipose tissue derived stem cells. Due to its proliferation inducing function and vast availability, this peptide is thus suitable for further application and investigation in various fields such as tissue engineering and stem cell research.

Application of high-mobility-group-A proteins increases the proliferative activity of chondrocytes in vitro.

The low capability of self-repair in hyaline cartilage tissue and chondrocytes de-differentiating when grown in vitro (e.g., for tissue engineering approaches) limits articular cartilage repair. It has been shown that the embryonic architectural transcription factors of the high-mobility-group-A (HMGA) protein family affect the regulation of cell differentiation by influencing the state of cell chromatin and are involved in hyaline cartilage development by affecting the expression of chondrocyte-specific marker genes. Thus, the control of cartilage cell proliferation and differentiation by HMGA proteins promises to be an important aspect in cartilage tissue repair. To elucidate the effects on the proliferative activity of hyaline chondrocytes, HMGA proteins were recombinantly expressed, highly purified, and applied to porcine hyaline cartilage cells growing in in vitro monolayer cell culture. Direct application of HMGA1a, HMGA1b, and HMGA2 proteins onto porcine chondrocytes was shown to have a highly significant influence on cell proliferation. Greater proliferation of chondrocytes was achieved than in the untreated control group, indicating a promising approach to enhancing cartilage tissue repair.

Cellular methods in cartilage research: primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells.

Work in our laboratory has focused on the in vitro culture of both human articular chondrocytes and human mesenchymal stem cells to understand what controls their ability to synthesise an appropriate cartilage-like extracellular matrix containing a predominantly collagen type II fibrillar network embedded in an aggrecan-rich ECM. This review focuses on the methodologies that we have found to be successful with cartilage and bone marrow sources of human cells and comments on the many factors which may enable improved phenotypic performance once the cells are in a fully chondrogenic environment.

HMGB1 protein inhibits DNA replication in vitro: a role of the acetylation and the acidic tail.

The high mobility group box (HMGB) 1 protein is a very abundant and conserved protein that is implicated in many key cellular events but its functions within the nucleus remain elusive. The role of this protein in replication of closed circular DNA containing a eukaryotic origin of replication has been studied in vitro by using native and recombinant HMGB1 as well as various modified HMGB1 preparations such as truncated protein, lacking its C-terminal tail, in vivo acetylated protein, and recombinant HMGB1 phosphorylated in vitro by protein kinase C (PKC). Native HMGB1 extracted from tumour cells inhibits replication and this effect is reduced upon acetylation and completely abolished upon removal of the acidic C-terminal tail. Recombinant HMGB1, however, fails to inhibit replication but it acquires such a property following in vitro phosphorylation by PKC.

Transcriptional regulation of scar gene expression in primary astrocytes.

The failure of the adult injured spinal cord to support axonal regeneration is in part attributed to the glial scar. Reactive astrocytes constitute a major cellular component of the glial scar and are heterogeneous with respect to the extracellular matrix proteins that they secrete. Astrocytes may produce antiregenerative molecules such as chondroitin sulphate proteoglycans (CSPGs) or proregenerative molecules such as laminin and fibronectin. While many different CSPGs are expressed after spinal cord injury (SCI) they all rely on the same enzymes, xylosyltransferase-I and -II (XT-I, XT-II) and chondroitin 4-sulfotransferase (C4ST) to add the repulsive chondroitin sulfate side chains to their core proteins. We show that XT-I, XT-II, and C4ST are part of a CSPG biosynthetic gene (CBG) battery. Using primary astrocyte cultures and quantitative PCR we demonstrate that TGFbeta2, PDGF, and IL-6 induce the expression of CBGs, laminin and fibronectin by several-fold. We further show that over-expression of the transcription factor SOX9 also strongly induces the expression of CBGs but does not increase the expression of laminin or fibronectin. Correspondingly, SOX9 knock-down in primary astrocytes causes a decrease in CBG and an increase in laminin and fibronectin mRNA levels. Finally, we show that the in vivo expression profiles of TGFbeta2, PDGF, IL-6, and SOX9 are consistent with their potential roles in differentially regulating CBGs, laminin and fibronectin gene expression in the injured spinal cord. This work suggests that SOX9 levels may be pivotal in determining the balance of pro- and anti-regenerative extracellular matrix molecules produced by astrocytes.

High-mobility group box 1 protein promotes development of microvascular thrombosis in rats.

BACKGROUND: Sepsis is a life-threatening disorder resulting from systemic inflammatory and coagulatory responses to infection. High-mobility group box 1 protein (HMGB1), an abundant intranuclear protein, was recently identified as a potent lethal mediator of sepsis. However, the precise mechanisms by which HMGB1 exerts its lethal effects in sepsis have yet to be confirmed. We recently reported that plasma HMGB1 levels correlated with disseminated intravascular coagulation (DIC) score, indicating that HMGB1 might play an important role in the pathogenesis of DIC. OBJECTIVES: To investigate the mechanisms responsible for the lethal effects of HMGB1, and more specifically, to explore the effects of HMGB1 on the coagulation system. METHODS: Rats were exposed to thrombin with or without HMGB1, and a survival analysis, pathologic analyses and blood tests were conducted. The effects of HMGB1 on the coagulation cascade, anticoagulant pathways and surface expression of procoagulant or anticoagulant molecules were examined in vitro. RESULTS: Compared to thrombin alone, combined administration of thrombin and HMGB1 resulted in excessive fibrin deposition in glomeruli, prolonged plasma clotting times, and increased mortality. In vitro, HMGB1 did not affect clotting times, but inhibited the anticoagulant protein C pathway mediated by the thrombin-thrombomodulin complex, and stimulated tissue factor expression on monocytes. CONCLUSIONS: These findings demonstrate the procoagulant role of HMGB1 in vivo and in vitro. During sepsis, massive accumulation of HMGB1 in the systemic circulation would promote the development of DIC.

Comparative effects of bone morphogenetic proteins and sox9 overexpression on extracellular matrix metabolism of bovine nucleus pulposus cells.

STUDY DESIGN: An in vitro biologic study of the effects of adenovirus expressing bone morphogenetic proteins (BMPs) and adenovirus expressing Sox9 on extracellular matrix metabolism by bovine nucleus pulposus cells. OBJECTIVE: To compare the effects of recombinant adenoviral vectors expressing various BMPs (2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15) and Sox9 on extracellular matrix accumulation by bovine nucleus pulposus cells. SUMMARY OF BACKGROUND DATA: Nucleus pulposus matrix production may be promoted by transducing the cells with genes that permit the sustained expression of growth factors. The choice of the particular factors or BMPs to be studied for these applications has been largely based on the commercial availability of such products. To our knowledge, this study is the first effort to evaluate systematically the relative effectiveness of the various members of the BMP family in promoting intervertebral disc matrix repair. METHODS: Adult bovine nucleus pulposus cells cultured in monolayer were transduced with adenoviruses expressing human BMP-2, 3, 4, 5, 7, 8, 10, 11, 12, 13, 14, and 15, and adenovirus expressing Sox9. Proteoglycan and collagen accumulation, and cell proliferation were measured 6 days after viral transduction. As a positive control, cells were cultured without any exogenous gene in the presence of recombinant human (rh)BMP-7. RESULTS: Nucleus pulposus cells transduced with adenoviruses expressing BMP-2, 3, 4, 5, 7, 8, 10, 13, 15, and Sox9 accumulated more proteoglycans than nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein (control). It is noteworthy that nucleus pulposus cells transduced with adenoviruses expressing BMP-2 and 7 resulted in essentially as great a stimulation of proteoglycan accumulation as nucleus pulposus cells maintained in the presence of rhBMP-7 (adenoviruses expressing BMP-2: 104% increase; adenoviruses expressing BMP-7: 162% increase; and rhBMP-7: 120% increase). Nucleus pulposus cells transduced with BMP-2, 4, 5, 7, 8, 10, 14, 15, and Sox9 accumulated significantly more collagen compared to nucleus pulposus cells transduced with adenovirus expressing green fluorescent protein; adenoviruses expressing BMP-4 and 14 were the most effective (552% and 661% increase, respectively). Nucleus pulposus cells also proliferated, as measured by deoxyribonucleic acid content, when transduced with adenoviruses expressing BMP-2 and 8. CONCLUSIONS: To our knowledge, for the first time, we have shown the relative effectiveness of 12 different BMPs and Sox9 in stimulating proteoglycan and collagen production by nucleus pulposus cells. Adenoviruses expressing BMP-2 and 7 were the most effective in stimulating proteoglycan accumulation, while adenoviruses expressing BMP-4 and 14 were the most effective in stimulating collagen accumulation. To our knowledge, this study is the first to compare the relative effectiveness of various BMPs and Sox9 on extracellular matrix accumulation by nucleus pulposus cells, and could help to develop more efficacious approaches to the treatment of degenerating intervertebral discs.

The high mobility group box chromosomal protein 1 is expressed in the human and rat testis where it may function as an antibacterial factor.

BACKGROUND: The high mobility group box chromosomal protein 1 (HMGB1) was originally shown to be a nuclear DNA-binding protein that activates transcription and promotes differentiation. More recently, there have been reports that HMGB1 may also function as a pro-inflammatory and antibacterial factor. The aim of this study was to investigate the testicular expression and antibacterial functions of HMGB1 to elucidate a possible role of HMGB1 in the testicular barrier defence against infections. METHODS AND RESULTS: RT-PCR and in situ hybridization revealed high-level testicular expression of HMGB1 mRNA and localization of this expression to the Sertoli cells and germ cells of the human and rat testis. In addition, immunohistochemical examination demonstrated the presence of the corresponding protein in Sertoli cells and spermatogonia in human and rat testes. Western blotting detected abundant amounts of the HMGB1 protein in the interstitial and intratubular fluids of the intact adult rat testis. Finally, the HMGB1 protein purified from both human and rat testis by reversed-phase high-performance liquid chromatography (HPLC) exerted antibacterial activity towards Bacillus megaterium in an inhibition zone assay. CONCLUSION: HMGB1 is expressed by Sertoli cells and germ cells in the mammalian testis. In addition, purified testicular HMGB1 shows antibacterial activity, indicating that this protein may function as a paracrine host defence factor in the testis.

HMGB1, a novel cytokine-like mediator linking acute neuronal death and delayed neuroinflammation in the postischemic brain.

Cerebral ischemic injury proceeds with excitotoxicity-induced acute neuronal death in the ischemic core and with delayed damage processes in the penumbra. However, knowledge concerning the direct mediators that connect these two processes is limited. Here, we demonstrate that high-mobility group box 1 (HMGB1), a nonhistone DNA-binding protein, is massively released into the extracellular space immediately after ischemic insult and that it subsequently induces neuroinflammation in the postischemic brain. Short hairpin (sh)RNA-mediated HMGB1 downregulation in the postischemic brain suppressed infarct size, microglia activation, and proinflammatory marker induction, indicating that HMGB1 plays a crucial role in the inflammatory process. The proinflammatory cytokine-like function of extracellular HMGB1 was further verified in primary cortical cultures and microglial cultures. HMGB1 was found to accumulate in NMDA-treated primary cortical culture media, and supernatants collected from these cultures were found to trigger microglia activation, the hallmark of brain inflammation. Moreover, treatment with recombinant HMGB1 also induced microglial activation, but HMGB1-depleted supernatant produced by anti-HMGB1 antibody treatment or by HMGB1 shRNA expression did not, thus demonstrating the essential role of HMGB1 in microglial activation. Together, these results indicate that HMGB1 functions as a novel proinflammatory cytokine-like factor that connects excitotoxicity-induced acute damage processes and delayed inflammatory processes in the postischemic brain.

SoxE factors function equivalently during neural crest and inner ear development and their activity is regulated by SUMOylation.

Sox9 and the closely related factor Sox10 are essential for the formation of neural crest precursor cells, and play divergent roles in the process by which these cells are subsequently directed to form specific derivatives. These group E Sox factors have also been implicated in the development of the vertebrate inner ear. Despite their importance, however, the mechanisms that allow SoxE proteins to regulate such a diverse range of cell types have remained poorly understood. Here we demonstrate that during vertebrate development, the activities of individual SoxE factors are well conserved and are regulated by SUMOylation. We show that SoxE mutants that cannot be SUMOylated, or that mimic constitutive SUMOylation, are each able to mediate a subset of the diverse activities characteristic of wild-type SoxE proteins. These findings provide important mechanistic insight into how the activity of widely deployed developmental regulatory proteins can be directed to specific developmental events.

RAGE is the major receptor for the proinflammatory activity of HMGB1 in rodent macrophages.

Abstract High-mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine-like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mphi) and define pathways activated by HMGB1 binding. Bone marrow Mphi were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK- and SAPK/JNK-signalling pathways, nuclear translocation of nuclear factor kappa B (NF-kappaB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mphi from interleukin (IL)-1 receptor type I-/-, Toll-like receptor 2 (TLR2-/-) and RAGE-/- mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mphi, phosphorylation of all investigated MAP kinase pathways and NF-kappaB translocation, and expression of MHC class II was increased. Mphi from RAGE-/- mice produced significantly lower amounts of TNF, IL-1beta and IL-6, while IL-1RI-/- and TLR2-/- Mphi produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mphi, with RAGE as the major activation-inducing receptor.

The combination of SOX5, SOX6, and SOX9 (the SOX trio) provides signals sufficient for induction of permanent cartilage.

OBJECTIVE: To regenerate permanent cartilage, it is crucial to know not only the necessary conditions for chondrogenesis, but also the sufficient conditions. The objective of this study was to determine the signal sufficient for chondrogenesis. METHODS: Embryonic stem cells that had been engineered to fluoresce upon chondrocyte differentiation were treated with combinations of factors necessary for chondrogenesis, and chondrocyte differentiation was detected as fluorescence. We screened for the combination that could induce fluorescence within 3 days. Then, primary mesenchymal stem cells, nonchondrogenic immortalized cell lines, and primary dermal fibroblasts were treated with the combination, and the induction of chondrocyte differentiation was assessed by detecting the expression of the cartilage marker genes and the accumulation of proteoglycan-rich matrix. The effects of monolayer, spheroid, and 3-dimensional culture systems on induction by combinations of transcription factors were compared. The effects of the combination on hypertrophic and osteoblastic differentiation were evaluated by detecting the expression of the characteristic marker genes. RESULTS: No single factor induced fluorescence. Among various combinations examined, only the SOX5, SOX6, and SOX9 combination (the SOX trio) induced fluorescence within 3 days. The SOX trio successfully induced chondrocyte differentiation in all cell types tested, including nonchondrogenic types, and the induction occurred regardless of the culture system used. Contrary to the conventional chondrogenic techniques, the SOX trio suppressed hypertrophic and osteogenic differentiation at the same time. CONCLUSION: These data strongly suggest that the SOX trio provides signals sufficient for the induction of permanent cartilage.

Induction of IL-10 in rat peritoneal macrophages and dendritic cells by glatiramer acetate.

Glatiramer acetate (GLAT) is a mixture of basic polypeptides that have been shown to suppress experimental autoimmune encephalomyelitis (EAE). As Copaxone, GLAT is approved for the treatment of relapsing-remitting multiple sclerosis (MS). Different immunomechanisms have been suggested to contribute to the beneficial effects of GLAT which rely on blockade of MHC class II molecules or cross-recognition with myelin basic protein (MBP). Because GLAT could also inhibit experimental autoimmunity not related to myelin proteins, we searched for additional, less-restricted immunomodulatory actions of GLAT. Using freshly isolated resident peritoneal macrophages from naive Lewis rats, it is shown that GLAT profoundly modulates cytokine secretion of the cells. In unseparated macrophages (MPhi) and MPhi of low density, GLAT enhanced constitutive and LPS-induced production of interleukin 10 (IL-10) while LPS-induced synthesis of tumor necrosis factor-alpha (TNF-alpha) was dose-dependently suppressed by GLAT. Although both basic proteins GLAT and MBP facilitated adherence of MPhi, MBP had opposite effects on cytokine production suggesting unique properties of GLAT. In contrast to MPhi, peritoneal mast cells produced only little amounts of cytokines. The inductive effect of GLAT on IL-10 production by antigen-presenting cells was also observed in bone marrow-derived rat dendritic cells (DCs) which, unlike MPhi, were not suppressed in their production of TNF-alpha. Induction of IL-10 in different antigen-presenting cells is a new immunomodulatory mechanism of GLAT. In part, it goes along with the inhibition of TNF-alpha and may be a common basis for the known beneficial effects of GLAT on various cellular autoimmune responses including MS.

Regulation of the orphan nuclear receptor steroidogenic factor 1 by Sox proteins.

Steroidogenic factor 1 (SF-1) is an essential factor in endocrine proliferation and gene expression. Despite the fact that SF-1 expression is restricted to specialized cells within the endocrine system, the only identified regulatory factors of SF-1 are the ubiquitously expressed E-box proteins (upstream stimulatory factors 1 and 2). Sequence examination of the SF-1 proximal promoter revealed a conserved site of AACAAAG (Sox-BS1), which matches exactly the defined consensus Sox protein binding element. Among the approximately 20 known members of the Sox gene family, we focused on Sox3, Sox8, and Sox9, based on their coexpression with SF-1 in the embryonic testis. Indeed, all three of these Sox proteins were capable of binding the proximal Sox-BS1 within the SF-1 promoter (-110 to -104), albeit with differing affinities. Of the three Sox proteins, Sox9 exhibited high-affinity binding to the Sox-BS1 element and consistently activated SF-1 promoter-reporter constructs. Mutating the Sox-BS1 attenuated SF-1 promoter activity in both embryonic and postnatal Sertoli cells, as well as in the adrenocortical cell line, Y1. Our findings, taken together with the overlapping expression profiles of Sox9 and SF-1, and the similar intersex phenotypes associated with both SOX9 and SF-1 human mutations, suggest that Sox9 up-regulates SF-1 and accounts partially for the sexually dimorphic expression pattern of SF-1 observed during male gonadal differentiation.

Cloning and characterization of the proximal murine Phex promoter.

Phex is an endopetidase that regulates systemic phosphate homeostasis. We investigated Phex gene transcription by cloning and performing functional analysis of the 2736 bp of the 5' flanking region of the mouse Phex gene containing its promoter. We identified a transcription start site, a consensus TATA-box, and multiple potential cis-acting regulator elements. To determine whether the promoter directs cell-type restricted Phex expression, we transfected full-length and 5'-deleted Phex luciferase reporter constructs into various cell lines. Phex-expressing C5.18 chondrocytes displayed the highest activity of the transfected Phex promoter constructs compared with non-Phex-expressing COS-7 cells, whereas promoter activity was intermediate in ROS 17/2.8 osteoblasts and maturation stage-dependent in MC3T3-E1 osteoblasts. Analysis of sequential 5'-deletion mutants of the Phex promoter in ROS 17/2.8 cells revealed bimodal activity, suggesting that both positive and negative cis-acting regions may be present. The chondrogenic factor SOX9 markedly stimulated Phex promoter activity, whereas Cbfa1, PTH, and 1,25(OH)(2)D(3) had no effect. Our findings are consistent with the predominant expression of Phex in bone and cartilage. Additional studies will be needed to confirm the regulatory regions in the Phex promoter that function in a cell-restricted manner.

Identity of nuclear high-mobility-group protein, HMG-1, and sulfoglucuronyl carbohydrate-binding protein, SBP-1, in brain.

High-mobility-group (HMG) proteins are a family of non-histone chromosomal proteins which bind to DNA. They have been implicated in multiple aspects of gene regulation and cellular differentiation. Sulfoglucuronyl carbohydrate binding protein, SBP-1, which is also localized in the neuronal nuclei, was shown to be required for neurite outgrowth and neuronal migration during development of the nervous system. In order to establish relationship between SBP-1 and HMG family proteins, two HMG proteins were isolated and purified from developing rat cerebellum by heparin-sepharose and sulfatide-octyl-sepharose affinity column chromatography and their biochemical and biological properties were compared with those of SBP-1. Characterization by high performance liquid chromatography--mass spectrometry (HPLC-MS), partial peptide sequencing and western blot analysis showed the isolated HMG proteins to be HMG-1 and HMG-2. Isoelectric focusing, HPLC-MS and peptide sequencing data also suggested that HMG-1 and SBP-1 were identical. Similar to SBP-1, both HMG proteins bound specifically to sulfated glycolipids, sulfoglucuronylglycolipids (SGGLs), sulfatide and seminolipid in HPTLC-immuno-overlay and solid-phase binding assays. The HMG proteins promoted neurite outgrowth in dissociated cerebellar cells, which was inhibited by SGGLs, anti-Leu7 hybridoma (HNK-1) and anti-SBP-1 peptide antibodies, similar to SBP-1. The proteins also promoted neurite outgrowth in explant cultures of cerebellum. The results showed that the cerebellar HMG-1 and -2 proteins have similar biochemical and biological properties and HMG-1 is most likely identical to SBP-1.

High mobility group 1 protein (HMG-1) stimulates proinflammatory cytokine synthesis in human monocytes.

Lipopolysaccharide (LPS) is lethal to animals because it activates cytokine release, causing septic shock and tissue injury. Early proinflammatory cytokines (e.g., tumor necrosis factor [TNF] and interleukin [IL]-1) released within the first few hours of endotoxemia stimulate mediator cascades that persist for days and can lead to death. High mobility group 1 protein (HMG-1), a ubiquitous DNA-binding protein, was recently identified as a "late" mediator of endotoxin lethality. Anti-HMG-1 antibodies neutralized the delayed increase in serum HMG-1, and protected against endotoxin lethality, even when passive immunization was delayed until after the early cytokine response. Here we examined whether HMG-1 might stimulate cytokine synthesis in human peripheral blood mononuclear cell cultures. Addition of purified recombinant HMG-1 to human monocyte cultures significantly stimulated the release of TNF, IL-1alpha, IL-1beta, IL-1RA, IL-6, IL-8, macrophage inflammatory protein (MIP)-1alpha, and MIP-1beta; but not IL-10 or IL-12. HMG-1 concentrations that activated monocytes were within the pathological range previously observed in endotoxemic animals, and in serum obtained from septic patients. HMG-1 failed to stimulate cytokine release in lymphocytes, indicating that cellular stimulation was specific. Cytokine release after HMG-1 stimulation was delayed and biphasic compared with LPS stimulation. Computer-assisted image analysis demonstrated that peak intensity of HMG-1-induced cellular TNF staining was comparable to that observed after maximal stimulation with LPS. Administration of HMG-1 to Balb/c mice significantly increased serum TNF levels in vivo. Together, these results indicate that, like other cytokine mediators of endotoxin lethality (e.g., TNF and IL-1), extracellular HMG-1 is a regulator of monocyte proinflammatory cytokine synthesis.

HMG1 protein stimulates DNA end joining by promoting association of DNA molecules via their ends.

High mobility group (HMG) 1 protein is a highly abundant and an evolutionarily conserved chromosomal protein with two homologous DNA-binding domains (HMG boxes), A and B, attached by a short basic region to an acidic C-terminal tail. The protein has been implicated in a number of fundamental biological processes including DNA replication, transcription, recombination and repair. We demonstrate that HMG1 is able to enhance cohesive-end and blunt-end DNA ligation by T4 DNA ligase via its B domain. The C-terminal flanking sequence of the B domain (seven basic residues out of approximately 18) and a number of conserved amino-acid residues within the HMG box (mainly basic or hydrophobic) are required for efficient stimulation of ligation. Pull-down assays, electron and scanning force microscopy revealed that HMG1 can associate two DNA molecules via their ends even in the absence of complementary overhangs. We propose that HMG1 protein may be involved in the rejoining of DNA breaks by different DNA ligases due to its ability to bring DNA duplexes and their termini into a close proximity while leaving the ends accessible for ligation.