Effects of low-intensity pulsed ultrasound on the infrapatellar fat pad in knee osteoarthritis: a randomized, double blind, placebo-controlled trial.

[Purpose] We investigated the effects of low-intensity pulsed ultrasound (LIPUS) irradiation of the infrapatellar fat pad (IFP) combined with therapeutic exercise for management of knee osteoarthritis (knee OA). [Participants and Methods] The study included 26 patients with knee OA, who were randomized into the LIPUS group (patients underwent LIPUS + therapeutic exercise) and the therapeutic exercise group (patients underwent sham LIPUS + therapeutic exercise). We measured changes in the patellar tendon-tibial angle (PTTA) and in IFP thickness, IFP gliding, and IFP echo intensity after 10 treatment sessions to determine the effects of the aforementioned interventions. We additionally recorded changes in the visual analog scale, Timed Up and Go Test, the Western Ontario and McMaster Universities Osteoarthritis Index, and Kujala scores, as well as range of motion in each group at the same end-point. [Results] Compared with patients in the therapeutic exercise group, those in the LIPUS group showed significant post-treatment improvements in PTTA, VAS, and Kujala scores, as well as in range of motion. [Conclusion] The combined use of LIPUS irradiation of the IFP and therapeutic exercise is a safe and effective modality to reduce IFP swelling, relieve pain, and improve function in patients with knee OA.

Influence of new coronavirus pandemic on behavior and awareness of young nurses and nursing students in Japan.

BACKGROUND: Although mental health disorders of health care workers in the coronavirus disease 2019 (COVID-19) pandemic have been focused, little is known about the psychological impact on nurses and the influence on their behavior and awareness, such as professionalism and views on life and death, in Japan where there are fewer cases of infection and deaths than in other countries. Moreover, the influence of the pandemic on nursing students is still unclear. METHODS: An online questionnaire survey was conducted among nurses and nursing students. Feelings during the state of emergency (at the peak of the pandemic) in Japan, changes in behavior and awareness after the rise of COVID-19, and the associated factors influencing these changes were analyzed, comparing nurses with nursing students. RESULTS: Significantly increased scores of anxiety/fear (p < .005) and voluntary restraint (p < .005) and significantly decreased score of motivation (p < .005) were observed during the state of emergency in both nurses and students. Scores of experience of discrimination (p < .005) and consideration of premature retirement (p < .01) were significantly increased in nurses. Moreover, preventive behavior (p < .005), lifestyle (p < .005), anxiety about nursing (p < .005) and views on life and death (p < .005) significantly changed after the rise of COVID-19 in both nurses and students. Only nurses reported significant damage to their professionalism (p < .01). Anxiety/fear and/or voluntary restraint and/or decreased motivation during the state of emergency were major factors associated with these changes. Also, the type of hospital, experience of care of infected patients and sex affected some of the changes. Voluntary restraint (p = .008), increased preventive behavior (p = .021) and decreased motivation (p = .005) were more marked in nurses than in students, while change in views on life and death was greater in students than in nurses (p = .002). CONCLUSION: The COVID-19 pandemic has had a psychological impact on nurses and nursing students, associated with changes in behavior and awareness even in Japan. Of note, the COVID-19 pandemic has affected nurses' professionalism and views on life and death. This study demonstrates the importance of having a coping strategy for anxiety and damaged professionalism in nurses, and education on life and death in nursing students.

Therapeutic effect of intra-articular injection of ribbon-type decoy oligonucleotides for hypoxia inducible factor-1 on joint contracture in an immobilized knee animal model.

BACKGROUND: Limited range of motion (ROM) as a result of joint contracture in treatment associated with joint immobilization or motor paralysis is a critical issue. However, its molecular mechanism has not been fully clarified and a therapeutic approach is not yet established. METHODS: In the present study, we investigated its molecular mechanism, focusing on the role of a transcription factor, hypoxia inducible factor-1 (HIF-1), which regulates the expression of connective tissue growth factor (CTGF) and vascular endothelial growth factor (VEGF), and evaluated the possibility of molecular therapy to inhibit HIF-1 activation by ribbon-type decoy oligonucleotides (ODNs) for HIF-1 using immobilized knee animal models. RESULTS: In a mouse model, ROM of the immobilized knee significantly decreased in a time-dependent manner, accompanied by synovial hypertrophy. Immunohistochemical studies suggested that CTGF and VEGF are implicated in synovial hypertrophy with fibrosis. CTGF and VEGF were up-regulated at both the mRNA and protein levels at 1 and 2 weeks after immobilization, subsequent to up-regulation of HIF-1 mRNA and transcriptional activation of HIF-1. Of importance, intra-articular transfection of decoy ODNs for HIF-1 in a rat model successfully inhibited transcriptional activation of HIF-1, followed by suppression of expression of CTGF and VEGF, resulting in attenuation of restricted ROM, whereas transfection of scrambled decoy ODNs did not. CONCLUSIONS: The present study demonstrates the important role of HIF-1 in the initial progression of immobilization-induced joint contracture, and indicates the possibility of molecular treatment to prevent the progression of joint contracture prior to intervention with physical therapy. Copyright © 2016 John Wiley & Sons, Ltd.

Wound healing potential of lavender oil by acceleration of granulation and wound contraction through induction of TGF-ß in a rat model.

BACKGROUND: Although previous studies have suggested that lavender oil promote wound healing, no study has examined the molecular mechanisms of its effect. In this study, we investigated the effect of lavender oil on various steps of wound healing and its molecular mechanism, focusing on transforming growth factor-ß (TGF-ß). METHODS: Circular full-thickness skin wounds were produced on rats. Control solution or lavender oil was topically applied to the wounds on alternating days for 14 days. RESULTS: The area of wounds topically treated with lavender oil was significantly decreased as compared to that of wounds of control rats at 4, 6, 8, and 10 days after wounding. Topical application of lavender oil induced expression of type I and III collagen at 4 days after wounding, accompanied by an increased number of fibroblasts, which synthesize collagen. Induced expression of type III collagen by topical application of lavender oil was reduced to control level at 7 days after wounding although increased expression of type I collagen still continued even at 7 days, suggesting rapid collagen replacement from type III to type I in wounds treated with lavender oil. Importantly, expression of TGF-ß in wounds treated with lavender oil was significantly increased as compared to control. Moreover, an increased number of myofibroblasts was observed in wounds treated with lavender oil at 4 days after wounding, suggesting promotion of differentiation of fibroblasts through induction of TGF-ß, which is needed for wound contraction. CONCLUSION: This study demonstrated that topical application of lavender oil promoted collagen synthesis and differentiation of fibroblasts, accompanied by up-regulation of TGF-ß. These data suggest that lavender oil has the potential to promote wound healing in the early phase by acceleration of formation of granulation tissue, tissue remodeling by collagen replacement and wound contraction through up-regulation of TGF-ß. The beneficial effect of lavender oil on wound healing may raise the possibility of new approaches as complementary treatment besides conventional therapy.

Effect of Low-Intensity Pulsed Ultrasound on Macrophage Properties and Fibrosis in the Infrapatellar Fat Pad in a Carrageenan-Induced Knee Osteoarthritis Rat Model.

BACKGROUND: In the progression of knee osteoarthritis (KOA), fibrosis of the infrapatellar fat pad (IFP) is a key pathological change. Low-intensity pulsed ultrasound (LIPUS) inhibits IFP fibrosis by decreasing the gene expression and activity of hypoxia-inducible factor (HIF-1α), which is a protein involved in IFP fibrosis in KOA rat models. On the other hand, macrophages play an important role in the progression of fibrosis in various tissues, and LIPUS irradiation suppresses macrophage infiltration and inflammatory cytokine secretion. However, whether LIPUS suppresses macrophage polarity and IFP fibrosis in KOA remains unclear. Therefore, we investigated the effect of LIPUS on macrophage polarity and IFP fibrosis. MATERIALS AND METHODS: A KOA model was created by injecting carrageenin into the bilateral knee joints of Wistar rats (eight weeks old). Tissues were harvested over time for histological and molecular biological analysis. The KOA model was also subjected to LIPUS irradiation for two weeks following the injection of carrageenin. RESULTS: RM-4-positive cells were widely distributed in IFP two weeks after carrageenin administration, but M2 macrophages were significantly increased, and the Sirius red area was decreased in the LIPUS-irradiated group compared with those in the non-irradiated group. The gene expression of M1 macrophage markers was significantly decreased and that of M2 macrophage markers was significantly increased in the LIPUS-irradiated group. The expression of transforming growth factor-ß (TGF-ß) and type 1 collagen was also significantly decreased. CONCLUSION: These results suggest that LIPUS may serve as a novel approach for the treatment of KOA through its effect on M1 macrophages and suppression of TGF-ß expression.

The influence of excessive IL-6 production in vivo on the development and function of Foxp3+ regulatory T cells.

IL-6 is a proinflammatory cytokine and its overproduction is implicated in a variety of inflammatory disorders. Recent in vitro analyses suggest that IL-6 is a key cytokine that determines the balance between Foxp3(+) regulatory T cells (Tregs) and Th17 cells. However, it remains unclear whether excessive IL-6 production in vivo alters the development and function of Foxp3(+) Tregs. In this study, we analyzed IL-6 transgenic (Tg) mice in which serum IL-6 levels are constitutively elevated. Interestingly, in IL-6 Tg mice, whereas peripheral lymphoid organs were enlarged, and T cells exhibited activated phenotype, Tregs were not reduced but rather increased compared with wild-type mice. In addition, Tregs from Tg mice normally suppressed proliferation of naive T cells in vitro. Furthermore, Tregs cotransferred with naive CD4 T cells into SCID-IL-6 Tg mice inhibited colitis as successfully as those transferred into control SCID mice. These results indicate that overproduction of IL-6 does not inhibit development or function of Foxp3(+) Tregs in vivo. However, when naive CD4 T cells alone were transferred, Foxp3(+) Tregs retrieved from SCID-IL-6 Tg mice were reduced compared with SCID mice. Moreover, the Helios(-) subpopulation of Foxp3(+) Tregs, recently defined as extrathymic Tregs, was significantly reduced in IL-6 Tg mice compared with wild-type mice. Collectively, these results suggest that IL-6 overproduced in vivo inhibits inducible Treg generation from naive T cells, but does not affect the development and function of natural Tregs.

Inhibitory effect of low­intensity pulsed ultrasound on the fibrosis of the infrapatellar fat pad through the regulation of HIF­1α in a carrageenan­induced knee osteoarthritis rat model.

Fibrotic changes in the infrapatellar fat pad (IFP) are involved in the pathogenesis of knee osteoarthritis (KOA). HIF-1α is a transcription factor that is activated during hypoxia and is suggested to play a role in fibrosis in various organs. However, its participation in the fibrotic changes in IFP remains unclear. Therefore, we investigated the role of HIF-1α in IFP fibrosis using a carrageenan-induced KOA rat model and evaluated the potential of low-intensity pulsed ultrasound (LIPUS) as a novel treatment for KOA. A rat model was prepared by intra-articular injection of 0.5% carrageenan (50 µl) using 8-week-old male Wistar rats. Fibrosis of the IFP was evaluated histologically by hematoxylin and eosin and Sirius Red staining at 1 and 2 weeks after intra-articular injection. The mRNA expression levels of HIF-1α and fibrosis-related molecules, CTGF and VEGF, were analyzed using reverse transcription-quantitative PCR, and the DNA binding activity of HIF-1α was assessed using a binding assay. In addition, the effect of irradiation with LIPUS on the fibrosis of IFP was verified. Histological studies demonstrated a significant increase in the fibrosis of IFP 1 and 2 weeks after intra-articular injection of carrageenan, accompanied by overexpression of CTGF and VEGF, which was followed by upregulation of transcriptional activation of HIF-1α. Moreover, intervention with LIPUS for 2 weeks after injection of carrageenan attenuated fibrosis of IFP, accompanied by a significant reduction in the transcriptional activation of HIF-1α and decreased the gene expression levels of HIF-1α, CTGF, and VEGF. The present study demonstrated that activation of HIF-1α promoted fibrosis of IFP in carrageenan-induced arthritis in rats and that intervention with LIPUS decreased the activity of HIF-1α and inhibited fibrosis. These results suggest that LIPUS may serve as a novel approach for the treatment of KOA, through its modulation of HIF-1α.

Blockade of interleukin-6 signaling suppresses experimental autoimmune uveoretinitis by the inhibition of inflammatory Th17 responses.

The aim of this study was to investigate the effect of anti-mouse IL-6 receptor monoclonal antibody (MR16-1) treatment on CD4 T cell differentiation and compared it to the effect of anti-TNF mAb treatment with using a murine model of experimental autoimmune uveoretinitis (EAU). C57BL/6 mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) to induce ocular inflammation treatment with control IgG or MR16-1 or anti-TNF mAb. Helper T cells differentiation was analyzed during the development of EAU. Immunization with IRBP increased the frequency of Th17 cells rather than Th1 cells in the early stage of EAU. Treatment with MR16-1 on the same day as immunization (day 0) or one day after (day 1) suppressed ocular inflammation in EAU mice. Treatment with MR16-1 on day 0 inhibited the induction of Th17 cells in vivo, and inhibited not only IRBP-responsive Th17 cells but also their Th1 counterparts and induced IRBP-responsive regulatory T (Treg) cells in vitro. The administration of anti-TNF mAb had no significant protective effect in EAU mice. The protective effect of anti-IL-6R mAb treatment, but not anti-TNF mAb treatment on EAU correlated with the inhibition of Th17 differentiation. This finding suggests that IL-6 blockade may have a therapeutic effect on human ocular inflammation which is mediated via mechanisms distinct from those of TNF blockade. IL-6 blockade may thus represent an alternative therapy for patients with ocular inflammation who are refractory to anti-TNF mAb therapy.

Moxibustion Promotes Formation of Granulation in Wound Healing Process through Induction of Transforming Growth Factor-ß in Rats.

OBJECTIVE: To examine the effect of moxibustion on the wound healing process and its mechanism using a rat wound model. METHODS: Sixty male Sprague-Dawley rats were randomly divided into a sham-treated group (n=30, wound surgery only) and a moxibustion group (n=30, wound treated with moxibustion). Circular full-thickness skin wounds were produced in rats. Moxibustion was applied to the edge of wound and was continued on alternating days till 14 days after surgery, followed by measurement of wound size. Expression of collagens, prolyl-4-hydroxylase (P4H) and transforming growth factor-ß (TGF-ß) were evaluated by histochemical study and real-time polymerase chain reaction. RESULTS: The size of the wound lesion was significantly reduced in rats treated with moxibustion as compared to that in sham-treated rats at 4-10 days after wounding (P<0.01). Moxibustion stimulated mRNA expression of collagens at 4 days (P<0.01), but not at 7 days, accompanied by enhanced proliferation of P4H-positive fibroblasts. Of importance, expression of TGF-ß in tissue from the wound lesion treated with moxibustion was significantly increased as compared to that in sham-treated rats at 4 days (P<0.01 or P<0.05), but not at 7 days. CONCLUSIONS: The treatment with moxibustion promoted the wound healing process in the early phase through proliferation of fibroblasts and rapid formation of granulation, possibly mediated by induction of TGF-ß which is a key molecule in the physiological process of wound healing. Moxibustion can be expected to be effective as complementary treatment for intractable ulcers.

Beneficial effect of laughter therapy on physiological and psychological function in elders.

AIM: In the present study we investigated the effect of laughter therapy on physiological and psychological function in older people. DESIGN: An open-label trial. METHODS: Seventeen older people who regularly attended an elderly day care centre were recruited. Stand-up comedy as laughter therapy was performed once a week for 4 weeks. Parameters of physiological and psychological function were evaluated before and after laughter therapy. RESULTS: Laughter therapy intervention resulted in a significant reduction in systolic blood pressure and heart rate, accompanied by a significant increase in plasma concentration of serotonin and a significant decrease in salivary concentration of chromogranin A. Questionnaire surveys of SF-8, GDS-15, and Vitality Index demonstrated alleviation of depression and improvement of sociability and activity in older people. Laughter therapy could be expected to become a practical treatment to improve quality of life of older people in an elderly day care centre.

Leucine-rich alpha 2 glycoprotein promotes Th17 differentiation and collagen-induced arthritis in mice through enhancement of TGF-ß-Smad2 signaling in naïve helper T cells.

BACKGROUND: Leucine-rich alpha 2 glycoprotein (LRG) has been identified as a serum protein elevated in patients with active rheumatoid arthritis (RA). Although the function of LRG is ill-defined, LRG binds with transforming growth factor (TGF)-ß and enhances Smad2 phosphorylation. Considering that the imbalance between T helper 17 (Th17) cells and regulatory T cells (Treg) plays important roles in the pathogenesis of RA, LRG may affect arthritic pathology by enhancing the TGF-ß-Smad2 pathway that is pivotal for both Treg and Th17 differentiation. The purpose of this study was to explore the contribution of LRG to the pathogenesis of arthritis, with a focus on the role of LRG in T cell differentiation. METHODS: The differentiation of CD4 T cells and the development of collagen-induced arthritis (CIA) were examined in wild-type mice and LRG knockout (KO) mice. To examine the influence of LRG on T cell differentiation, naïve CD4 T cells were isolated from LRG KO mice and cultured under Treg- or Th17-polarization condition in the absence or presence of recombinant LRG. RESULTS: In the CIA model, LRG deficiency led to ameliorated arthritis and reduced Th17 differentiation with no influence on Treg differentiation. By addition of recombinant LRG, the expression of IL-6 receptor (IL-6R) was enhanced through TGF-ß-Smad2 signaling. In LRG KO mice, the IL-6R expression and IL-6-STAT3 signaling was attenuated in naïve CD4 T cells, compared to wild-type mice. CONCLUSIONS: Our findings suggest that LRG upregulates IL-6R expression in naïve CD4 T cells by the enhancement of TGF-ß-smad2 pathway and promote Th17 differentiation and arthritis development.

Continuous infusion of angiotensin II modulates hypertrophic differentiation and apoptosis of chondrocytes in cartilage formation in a fracture model mouse.

Although components of the renin-angiotensin system (RAS) are reported to be expressed in cultured chondrocytes and cartilage, little is known about the precise function of Angiotensin II (Ang II) in chondrocytes. In this study, we employed a rib fracture model mouse to investigate the effect of Ang II on chondrocytes. Ang II type 1 receptor (AT1R) was expressed in chondrocytes in the growth plate of mouse tibia. Continuous infusion of Ang II to rib-fractured mice resulted in a significant increase in the volume of cartilage, suggesting Ang II-induced hypertrophic differentiation of chondrocytes. It was also confirmed by a significant increase in the mRNA expression of Sox9 and runt-related transcription factor 2 (Runx2), which are genes related to chondrocyte differentiation, and type X collagen, matrix metalloproteinase (MMP)-13 and Indian hedgehog (Ihh), which are hypertrophic chondrocyte-specific molecular markers. Chondrocyte hypertrophy with upregulation of these genes was attenuated by administration of olmesartan, an AT1R blocker, but not by hydralazine. Moreover, Ang II infusion significantly suppressed apoptosis of chondrocytes, accompanied by significant induction of mRNA expression of bcl-2 and bcl-xL. Olmesartan, but not hydralazine, significantly attenuated the reduction of apoptotic cells and the increase in anti-apoptotic genes induced by Ang II infusion. Overall, the present study demonstrated that Ang II promoted hypertrophic differentiation of chondrocytes and reduced apoptosis of hypertrophic chondrocytes independently of high blood pressure. The present data indicate the role of Ang II in cartilage, and might provide a new concept for treatment of cartilage diseases.

Effect of angiotensin II receptor blocker, olmesartan, on turnover of bone metabolism in bedridden elderly hypertensive women with disuse syndrome.

AIMS: Although recent studies suggest that several antihypertensive drugs could reduce the risk of bone fracture, it is still unclear how these drugs act on bone remodeling, especially in elderly women with severe osteoporosis with disuse syndrome. In the present study, we investigated the effects of a calcium channel blocker (CCB) and an angiotensin II receptor blocker (ARB) on bone metabolism in elderly bedridden women with hypertension and disuse syndrome. METHODS: Elderly bedridden women (aged >75 years) receiving antihypertensive therapy treated with CCB were recruited in the present study. The participants were divided into two groups--CCB group and ARB group--and followed up to 12 months. RESULTS: Markers of bone resorption were markedly increased, suggesting accelerated bone resorption in the participants of the present study. In the follow-up period, the patients treated with a CCB showed a significant decrease in bone mineral density in a time-dependent manner, accompanied by a significant increase in bone resorption markers, whereas treatment with olmesartan inhibited bone loss, associated with attenuation of increased bone resorption markers. Bone mineral density of femoral neck in the CCB group was significantly lower than that in the ARB group at 6 months. CONCLUSION: The present study showed inhibitory effects of an ARB on bone resorption in hypertensive patients with accelerated bone resorption, such as elderly bedridden women, and indicated an important role of the renin-angiotensin system in bone metabolism. In elderly hypertensive patients, ARB might be expected to have additional beneficial potential to maintain bone health in bedridden patients.

Identification of promoter regions involved in cell- and developmental stage-specific osteopontin expression in bone, kidney, placenta, and mammary gland: an analysis of transgenic mice.

UNLABELLED: Cell-specific expression of GFP under the control of different lengths of the osteopontin promoter in transgenic mice identified the positive and negative regulatory regions for respective cell types. The results provide new insights for physiological and pathological expression of the osteopontin gene. INTRODUCTION: Osteopontin (OPN) is a major non-collagenous bone matrix protein that is involved in normal and pathological calcification and is expressed in a tissue-specific manner. To investigate how such tissue-specific OPN gene expression is regulated in vivo, transgenic mice expressing the green fluorescent protein (GFP) reporter gene controlled by different lengths of the OPN promoter were generated. MATERIALS AND METHODS: Cell- and developmental stage-specific osteopontin expression in transgenic mice was examined by Northern blotting, immunoblotting, fluorescence examination, and in situ hybridization and compared with those of OPN. RESULTS AND CONCLUSIONS: The line bearing the -5505 to +14 region of the OPN promoter was shown by Northern blotting and immunoblotting to express GFP in the same cells that express endogenous OPN (osteoblasts, hypertrophic chondrocytes, renal and mammary gland epithelial cells, and granulated metrial gland [GMG] placental cells) at the same stage in development. Thus, the 5.5-kb -5505 to +14 promoter region is sufficient for proper tissue-specific OPN expression. The lines carrying shorter segments of the OPN promoter showed different expression patterns. These patterns revealed a putative cis-acting element in the -5269 to -5263 region that restricts OPN expression to hypertrophic chondrocytes and a mammary gland-specific expressing element and a GMG cell-specific enhancing element in the -5505 to -3156 region. Furthermore, the -3155 to -1576 region seems to contain positive renal epithelial cell- and GMG cell-specific expression motif(s) as well as a negative regulatory element that prevents OPN expression in fibroblasts. Moreover, the -1576 to -910 region seems to contain a positive osteoblast-specific-expressing element. Thus, the 5.5-kb OPN promoter contains multiple cis-acting elements encoding positive and negative cell-specific regulatory systems.

Comparative analysis of the effects of anti-IL-6 receptor mAb and anti-TNF mAb treatment on CD4+ T-cell responses in murine colitis.

BACKGROUND: The efficacy of anti-tumor necrosis factor monoclonal antibody (anti-TNF mAb) for Crohn's disease (CD) is well established, and anti-interleukin-6 receptor (anti-IL-6R) mAb has also been reported to be effective in CD. It is, however, unclear if the efficacy and mechanisms of both agents are different in CD therapy. METHODS: Using an adoptive transfer colitis model, we compared the efficacy of anti-IL-6R mAb, anti-TNF mAb, and TNF receptor-Fc fusion protein (TNFR-Fc), and their modes of action on CD4+ T cells. We also investigated the role of Th1 and Th17 cells in colitis using the same model. RESULTS: The histological scores for the anti-IL-6R mAb and anti-TNF mAb groups but not for TNFR-Fc group were much lower than that for the control group, and the score was the lowest for the anti-IL-6R mAb group. The frequency of proliferating CD4+ T cells was reduced in anti-IL-6R mAb and anti-TNF mAb groups, but not in the TNFR-Fc group, whereas the frequency of apoptotic CD4+ T cells was similar in all groups. Anti-IL-6R mAb suppressed the induction of Th17 cells and increased the frequency of lamina propria regulatory T cells, whereas anti-TNF mAb exerted no influence on CD4+ T-cell differentiation. A deficiency in interferon-γ and/or IL-17 in CD4+ T cells reduced the severity of colitis. CONCLUSIONS: Our findings suggest that suppression of the proliferation of pathogenic CD4+ T cells is the major mode of action of biological agents for colitis therapy. Anti-IL-6R mAb might have benefits in CD patients with Th17 dominance and impaired Treg frequency.

Difference of osteopontin gene regulation between bone and kidney.

Osteopontin is a sialoprotein that is expressed in various cells. It plays a variety of important roles in cell adhesion, migration, signaling, calcification, and immunity. Its diverse functions indicate that the regulation of osteopontin may also vary extensively among tissues. Although osteopontin promoter has been studied in vitro, in vivo analyses may be more appropriate for elucidating osteopontin's functions. In an attempt to investigate osteopontin gene expression, we generated transgenic mice in which the bacterial beta-galactosidase reporter gene was conjugated downstream of osteopontin promoter. The osteopontin promoter was a mouse -910 bp upstream fragment, which we had previously found functional in 3T3 cells. Among 34 transgenic founders, 13 mice were transgenic, as determined with the polymerase chain reaction. Osteopontin and beta-galactosidase signals were evaluated with in situ hybridization. Among the 13 transgenic mice, 3 were beta-galactosidase-positive. In these transgenic mice, osteopontin signals were observed in bones and kidneys, whereas beta-galactosidase message was detected only in bones. This suggests that the -910 bp osteopontin promoter is active in bones but not in kidneys. These data imply that the promoter region required for osteopontin expression in kidneys may differ from that in bones.

Enhanced expression of Runx2/PEBP2alphaA/CBFA1/AML3 during fracture healing.

The cause of the dramatic increase in expression of the osteopontin gene during fracture healing was studied in a mouse experimental model. Semiquantitative reverse transcription-polymerase chain reaction, Northern blotting, and in situ hybridization analysis showed that the enhanced expression took place prior to callus formation. The change in the expression pattern of collagenous and noncollagenous bone matrix proteins in addition to Ets-1 and Runx2, major transcription factors of osteopontin, were examined and compared to that of osteopontin. Although Ets-1 expression showed no significant change during fracture healing, enhanced expression of Runx2 corresponding to that of osteopontin was observed. Furthermore, in situ hybridization demonstrated that osteopontin-expressing cells also express the Runx2 gene. The results indicated the possibility that Runx2 is a major regulator of osteopontin during fracture healing.