Kalervo Väänänen - Academia.edu (original) (raw)

Papers by Kalervo Väänänen

BoneKEy reports, Mar 13, 2013

We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytoti... more We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytotic route from the ruffled border to the functional secretory domain (FSD) in bone-penetrating osteoclasts. Cells of rat or human origin were cultured on bovine bone slices and analyzed via confocal microscopy. Helix pomatia lectin binding indicated that transcytotic vesicles expose aberrant N-acetylgalactosamine glycoconjugates, which is associated with a poor prognosis for a range of metastasizing human adenocarcinomas. Transcytotic vesicles fuse with the autophagosomal compartments and represent raft concentrates. Furthermore, the results of a vertical vesicle analysis suggest that multiple vesicle populations arise from the ruffled border and that some of these vesicles undergo a maturation process along the transcytotic route. Finally, our data suggest that the targeting of these membrane pathways may be determined by a novel F-actin-containing and FSD-circumscribing molecular barrier.

Journal of Cellular Biochemistry, Jun 15, 1999

Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal wo... more Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal women. These compounds seem to inhibit bone resorption, but their anabolic effects have been less explored. In this study, bone marrow cultures were used to compare the effect of 17beta-estradiol (E2), and two triphenylethylene derivatives, tamoxifen (TAM), and FC1271a, and a benzothiophene derivative raloxifene (RAL) on differentiation of osteoblasts. All enhanced osteoblastic differentiation of 21-day cultures as indicated by increased mineralization and bone nodule formation. All, except RAL, stimulated cell proliferation during the first 6 days of the culture. However, in the presence of RAL the content of total protein was increased in 13-day cultures. SDS-PAGE and autoradiography of [14C]-proline labeled proteins revealed elevated level of the newly synthesized collagen type I. The pure antiestrogen ICI 182,780 abolished the increase of the specific activity of alkaline phosphatase by E2, TAM, and FC1271a but not the effect of RAL on protein synthesis. Our results show that E2 as well as TAM, FC1271a, and RAL stimulate bone formation in vitro but the mechanism of the anabolic action of RAL in bone clearly differs from that of E2, TAM, and FC1271a.

Springer eBooks, 2002

Since the beginning of pharmacological use of glucocorticoids it has been clear that they have se... more Since the beginning of pharmacological use of glucocorticoids it has been clear that they have serious adverse effects in bone. Chronic administration of glucocorticoids causes a loss of bone mass and pathological fractures, especially in the skeletal areas that are rich in trabecular bone. In addition to osteoporosis, large doses of glucocorticoids could lead to aseptic osteonecrosis affecting mainly proximal femur.

Calcified Tissue International, May 1, 1986

Rhodamine-conjugated Arachis hypogaea (peanut agglutinin, PNA) lectin, which is specific for 13-D... more Rhodamine-conjugated Arachis hypogaea (peanut agglutinin, PNA) lectin, which is specific for 13-D-galactosyl and [3-D-gal-[1,3]-D-galNAC residues, was used to identify osteoclasts in paraffin-embedded bone sections of fetal rat calvaria and a human bone-derived tumor, osteoclastoma. All multinucleated osteoclasts were positive for PNA-lectin. This was also confirmed by studying smears of isolated osteoclasts from rat and chicken long bones. In addition to multinuclear cells, some mononuclear cells on the endosteal surface of the rat calvaria and some bone marrow cells also revealed specific binding of PNA lectin. Isolated rat peripheral monocytes were also studied, and these showed specific binding of PNA lectin which was maintained unchanged for at least 3 days in culture. Different lectins could be useful as membrane markers for studying bone cell origin and maturation.

Endocrinology, Jun 1, 1991

Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostat... more Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Gene regulation and systems biology, 2008

MicroRNAs (miRNAs) are non-coding RNAs that bind to target mRNA leading to translational arrest o... more MicroRNAs (miRNAs) are non-coding RNAs that bind to target mRNA leading to translational arrest or mRNA degradation. To study miRNA-mediated regulation of osteogenesis and chondrogenesis, we compared the expression of 35 miRNAs in osteoblasts and chondroblasts derived from mouse marrow stromal cells (MSCs). Differentiation of MSCs resulted in up-or downregulation of several miRNAs, with miR-199a expression being over 10-fold higher in chondroblasts than in undifferentiated MSCs. In addition, miR-124a was strongly upregulated during chondrogenesis while the expression of miR-96 was substantially suppressed. A systems biological analysis of the potential miRNA target genes and their interaction networks was combined with promoter analysis. These studies link the differentially expressed miRNAs to collagen synthesis and hypoxia, key pathways related to bone and cartilage physiology. The global regulatory networks described here suggest for the fi rst time how miRNAs and transcription factors are capable of fi ne-tuning the osteogenic and chondrogenic differentiation of mouse MSCs.

Journal of Biomechanical Science and Engineering, 2007

Osteocyte is the most abundant cell type in bone, and the only cell type located inside the miner... more Osteocyte is the most abundant cell type in bone, and the only cell type located inside the mineralized matrix. The striking structural design of bone predicts an important role for osteocytes in determining bone structure and function. Osteocytes are connected with each other via gap junctions and form a three dimensional cellular network in mineralized bone matrix. Recently, it has been shown that osteocytes are not only passive bystanders, but also have an active regulatory role in whole body phosphate and calcium metabolism. Osteocytes are cells which sense mechanical loading in bone. They respond to mechanical stimuli by producing and secreting several bioactive substances including nitric oxide and prostaglandins and thus transmit messages of loading to effector cells, like osteoblasts and osteoclasts. Present data suggest that osteocytes actively inhibit osteoclastic bone resorption. Whenever osteocytes die, this inhibitory effect is turned off and osteoclasts are activated. This mechanism of action could explain targeted remodeling in the region of stress induced microcracks happened. Osteocytes can also modulate osteoblasts function. In conclusion, osteocytes sense mechanical stimuli, transmit signals through cellular network and regulate osteoblast and osteoclast function during bone remodeling.

Journal of Histochemistry and Cytochemistry, Oct 1, 1990

Bone resorbing osteodasts form a specific microfilament structure at the attachment area, in whic... more Bone resorbing osteodasts form a specific microfilament structure at the attachment area, in which vinculin and tabin appear as a doubbe-cirde structure and F-actin fills the space between these cirdes. This distribution of microfilaments is associated with the resorption lacunae, and F-actin, yinculin, and talin zones correspond roughly to the edges of the lacunae. In the present work, we examined by immunofluorescence the effects of calcitonin (CT) and prostaglandin E2 (PGE4, inhibitors of osteodastic activity, as well as dibutyryl cyclic AMP (Bt2cAMP) and cytochabasin B, on the microfilament organization in resorbing osteoclasts. CT, PGE2, and Bt2cAMP rapidly dispersed the specific microfilament structure in resorbing osteoclasts. All microfil-I Supported by a grant from Sigrid Jus#{233}lius Foundation and Paulo Foundation.

Journal of Cell Biology, Nov 15, 1991

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption ... more During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycinearginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed in-0 TEOC1.ASTs are multinucleated cells responsible for

12th European Congress of Endocrinology, Apr 1, 2010

Journal of Biomechanical Science and Engineering, 2007

Osteocyte is the most abundant cell type in bone, and the only cell type located inside the miner... more Osteocyte is the most abundant cell type in bone, and the only cell type located inside the mineralized matrix. The striking structural design of bone predicts an important role for osteocytes in determining bone structure and function. Osteocytes are connected with each other via gap junctions and form a three dimensional cellular network in mineralized bone matrix. Recently, it has been shown that osteocytes are not only passive bystanders, but also have an active regulatory role in whole body phosphate and calcium metabolism. Osteocytes are cells which sense mechanical loading in bone. They respond to mechanical stimuli by producing and secreting several bioactive substances including nitric oxide and prostaglandins and thus transmit messages of loading to effector cells, like osteoblasts and osteoclasts. Present data suggest that osteocytes actively inhibit osteoclastic bone resorption. Whenever osteocytes die, this inhibitory effect is turned off and osteoclasts are activated. This mechanism of action could explain targeted remodeling in the region of stress induced microcracks happened. Osteocytes can also modulate osteoblasts function. In conclusion, osteocytes sense mechanical stimuli, transmit signals through cellular network and regulate osteoblast and osteoclast function during bone remodeling.

BoneKEy Reports, 2013

We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytoti... more We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytotic route from the ruffled border to the functional secretory domain (FSD) in bone-penetrating osteoclasts. Cells of rat or human origin were cultured on bovine bone slices and analyzed via confocal microscopy. Helix pomatia lectin binding indicated that transcytotic vesicles expose aberrant N-acetylgalactosamine glycoconjugates, which is associated with a poor prognosis for a range of metastasizing human adenocarcinomas. Transcytotic vesicles fuse with the autophagosomal compartments and represent raft concentrates. Furthermore, the results of a vertical vesicle analysis suggest that multiple vesicle populations arise from the ruffled border and that some of these vesicles undergo a maturation process along the transcytotic route. Finally, our data suggest that the targeting of these membrane pathways may be determined by a novel F-actin-containing and FSD-circumscribing molecular barrier.

The Journal of cell biology, 1990

Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts ... more Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H(+)-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60- and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatment of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K(+)-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through ...

Journal of Histochemistry & Cytochemistry, 1989

The appearance of carbonic anhydrase isoenzymes in rat liver and skeletal muscle during fetal and... more The appearance of carbonic anhydrase isoenzymes in rat liver and skeletal muscle during fetal and postnatal development was studied with immunohistochemistry. In the 12-day fetus the early strong expression of CA I in hepatocytes was partially replaced by the expression of CA II and CA III during late prenatal development. In the 20-day fetus the staining intensity of CA III was equal to that of a mature female rat. In the male, the staining intensity in hepatocytes clearly increased during sexual maturation. Immunoelectron microscopy showed diffuse cytoplasmic and nucleoplasmic staining of CA III in hepatocytes. The time-dependent expression of the isoenzymes in hepatocytes may reflect a different metabolic function of these structurally closely related isoenzymes. In skeletal muscle, CA III was the only isoenzyme detected during development. In late prenatal and early postnatal stages all muscle fibers contained about equal amounts of CA III. The differentiation of the muscle acco...

Journal of Bone and Mineral Research, 2006

Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover ... more Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed an in vitro model, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Our results show that the damaged osteocytes activate the osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling. Introduction: Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Besides the targeted remodeling, some remodeling may also be random to serve metabolic purposes. Osteocytes have been considered to provide a crucial role in the activation of osteoclastic bone resorption adjacent to the damaged site. This study was aimed to develop a relevant in vitro model of the targeted remodeling and to show that damaged osteocytes can induce the initial bone resorptive stage. Materials and Methods: We developed a new device, in which osteocyte-like cell line MLO-Y4 cells were 3D cultured, subjected to local scratching, and assayed for cell viability. NIH3T3-3 cells were used as a control. Bone marrow cells were cultured on the top of the mechanically damaged MLO-Y4 cells, and the formation of TRACP + cells was assayed. Additionally, the conditioned medium from scratched cultures was added to bone marrow cultures, and the TRACP activity in cell lysates was quantified. The macrophage-colony stimulating factor (M-CSF) and RANKL secretion in the conditioned medium was assayed by ELISA. Results: Scratching induced the death of MLO-Y4 cells. When bone marrow cells were cultured over the gel-embedded MLO-Y4 cells, the application of mechanical scratching induced TRACP + cell differentiation on gel surface. The cells with TRACP + could be observed in the very restricted region along the scratching path. Additionally, mechanically damaged osteocytes secreted M-CSF and RANKL, and the conditioned medium showed the potential to induce TRACP + cells in bone marrow culture. Conclusions: These findings indicate that soluble factors secreted from damaged osteocytes can locally induce and activate the initial phase of osteoclastic cell formation. This study directly shows the association between the damaged osteocytes and the initiation of resorptive stage in bone remodeling.

Biochemical and Biophysical Research Communications, 2000

The Carbonic Anhydrase II (CAII) gene that encodes an enzyme involved in proton production is exp... more The Carbonic Anhydrase II (CAII) gene that encodes an enzyme involved in proton production is expressed in several cell types including monocyte/macrophagederived osteoclasts. We have analyzed the regulation of the chicken CAII promoter/reporter construct by nuclear hormone receptors of the VDR subfamily in HD11 avian macrophages. The CAII expression is stimulated by 1,25-dihydroxyvitamin D 3 but not by 9-cis retinoic acid and repressed by VDR overexpression due to RXR squelching. It is also stimulated by alltrans retinoic acid only when RAR␣ is overexpressed, and is dependent on a RARE located in the distal part of the promoter and bound by RAR␣ homodimer. Finally, in macrophages, unlike in erythrocytes, the CAII promoter is unresponsive to thyroid hormone. Our results demonstrate the first retinoic acid response element in the CAII promoter and show that according to cell type, different nuclear receptors of the VDR subfamily can regulate the CAII gene.

PLOS ONE, May 3, 2013

Objective: Osteocalcin (OC), a bone-derived protein, has been implicated in the regulation of glu... more Objective: Osteocalcin (OC), a bone-derived protein, has been implicated in the regulation of glucose and energy metabolism. Young adults born with very low birth weight (VLBW) have altered glucose regulation and lower bone mineral density (BMD) compared with those born at term. The aim of this study was to explore the association between bone and glucose metabolism in healthy young adults born prematurely or at term. Methods: The cohort of this cross-sectional study comprised 332 non-diabetic young adults (age 18 to 27 years) born either preterm with VLBW (n = 163) or at term (n = 169). OC, carboxylated osteocalcin (cOC) and markers of glucose metabolism were measured at fasting and after a 75-g oral glucose tolerance test (OGTT). Results: VLBW adults were shorter, had lower BMD (p,0.001) and higher fasting OC (p = 0.027) and cOC (p = 0.005) than term-born subjects. They also had higher 2-hour insulin (p = 0.001) and glucose (p = 0.037) concentrations. OGTT induced a significant reduction in OC (p,0.001), similar in both groups. OC reduction was not associated with OGTT-induced increases in insulin (p = 0.54). However, fasting total OC and cOC correlated negatively with fasting insulin after adjustment for age, gender, BMD and VLBW status (r = 20.182, p = 0.009 and r = 20.283, p,0.001, respectively). Conclusion: Adults born with VLBW have higher OC and cOC than their peers born at term. This may in part reflect the mechanisms that underlie their lower BMD and decreased insulin sensitivity. Serum OC appears to be negatively associated with long-term glucose regulation whereas acute changes during OGTT may be mediated via other mechanisms.

American Journal of Obstetrics and Gynecology, 2002

Objective: Estrogen replacement therapy after menopause reduces the incidence of arterial disease... more Objective: Estrogen replacement therapy after menopause reduces the incidence of arterial disease and cerebrovascular events. The reduced incidence also seems to be due to a positive effect of estrogens on brain blood flow as shown by a decrease in the carotid ...

BoneKEy reports, Mar 13, 2013

We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytoti... more We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytotic route from the ruffled border to the functional secretory domain (FSD) in bone-penetrating osteoclasts. Cells of rat or human origin were cultured on bovine bone slices and analyzed via confocal microscopy. Helix pomatia lectin binding indicated that transcytotic vesicles expose aberrant N-acetylgalactosamine glycoconjugates, which is associated with a poor prognosis for a range of metastasizing human adenocarcinomas. Transcytotic vesicles fuse with the autophagosomal compartments and represent raft concentrates. Furthermore, the results of a vertical vesicle analysis suggest that multiple vesicle populations arise from the ruffled border and that some of these vesicles undergo a maturation process along the transcytotic route. Finally, our data suggest that the targeting of these membrane pathways may be determined by a novel F-actin-containing and FSD-circumscribing molecular barrier.

Journal of Cellular Biochemistry, Jun 15, 1999

Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal wo... more Estrogens as well as some antiestrogens have been shown to prevent bone loss in postmenopausal women. These compounds seem to inhibit bone resorption, but their anabolic effects have been less explored. In this study, bone marrow cultures were used to compare the effect of 17beta-estradiol (E2), and two triphenylethylene derivatives, tamoxifen (TAM), and FC1271a, and a benzothiophene derivative raloxifene (RAL) on differentiation of osteoblasts. All enhanced osteoblastic differentiation of 21-day cultures as indicated by increased mineralization and bone nodule formation. All, except RAL, stimulated cell proliferation during the first 6 days of the culture. However, in the presence of RAL the content of total protein was increased in 13-day cultures. SDS-PAGE and autoradiography of [14C]-proline labeled proteins revealed elevated level of the newly synthesized collagen type I. The pure antiestrogen ICI 182,780 abolished the increase of the specific activity of alkaline phosphatase by E2, TAM, and FC1271a but not the effect of RAL on protein synthesis. Our results show that E2 as well as TAM, FC1271a, and RAL stimulate bone formation in vitro but the mechanism of the anabolic action of RAL in bone clearly differs from that of E2, TAM, and FC1271a.

Springer eBooks, 2002

Since the beginning of pharmacological use of glucocorticoids it has been clear that they have se... more Since the beginning of pharmacological use of glucocorticoids it has been clear that they have serious adverse effects in bone. Chronic administration of glucocorticoids causes a loss of bone mass and pathological fractures, especially in the skeletal areas that are rich in trabecular bone. In addition to osteoporosis, large doses of glucocorticoids could lead to aseptic osteonecrosis affecting mainly proximal femur.

Calcified Tissue International, May 1, 1986

Rhodamine-conjugated Arachis hypogaea (peanut agglutinin, PNA) lectin, which is specific for 13-D... more Rhodamine-conjugated Arachis hypogaea (peanut agglutinin, PNA) lectin, which is specific for 13-D-galactosyl and [3-D-gal-[1,3]-D-galNAC residues, was used to identify osteoclasts in paraffin-embedded bone sections of fetal rat calvaria and a human bone-derived tumor, osteoclastoma. All multinucleated osteoclasts were positive for PNA-lectin. This was also confirmed by studying smears of isolated osteoclasts from rat and chicken long bones. In addition to multinuclear cells, some mononuclear cells on the endosteal surface of the rat calvaria and some bone marrow cells also revealed specific binding of PNA lectin. Isolated rat peripheral monocytes were also studied, and these showed specific binding of PNA lectin which was maintained unchanged for at least 3 days in culture. Different lectins could be useful as membrane markers for studying bone cell origin and maturation.

Endocrinology, Jun 1, 1991

Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostat... more Hormone regulation of carbonic anhydrase II (CA II) was studied in rat dorsal and lateral prostate. CA II is a major soluble protein in these accessory sex glands. The immunoelectronmicroscopy showed that CA II is expressed in their epithelial cells only. For studies on hormone regulation, adult male rats were castrated for 2 or 7 days. Groups of 7-day castrates and normal rats were treated daily either with testosterone or 17-beta-estradiol for 6 days and 2-day castrates for 1 day. CA II protein was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and quantified by RIA. The levels of CA II mRNA were studied by Northern blotting and hybridization of total RNA with a 32P-labeled mouse CA II cDNA clone. Castration of the rats decreased the concentration of CA II in lateral prostate but increased in dorsal prostate. These changes were reversed in both prostatic lobes by testosterone treatment. Estrogen treatment of castrated rats enhanced CA II concentration in lateral prostate but no effects were seen in the dorsal prostate of the same animals. In normal rats estrogen increased CA II concentration of dorsal prostate but there was no change in lateral prostate. Corresponding changes were observed in the levels of CA II mRNA in both tissues. The morphometric analyses showed that the castration- and hormone-induced changes of the mRNA and protein levels of the exclusively epithelial CA II could not be explained by any alterations in the proportions of epithelial and stromal components of the glands after hormone manipulations. The results demonstrate the differential steroid regulation of CAII in two prostatic lobes. Androgen regulates the expression of CAII at messenger RNA level, but the responses of CAII to testosterone are opposite in dorsal and lateral prostate. Estrogen increases CA II expression in lateral prostate but in dorsal prostate the castration-like effects of estrogen on CAII expression are probably indirect.

Gene regulation and systems biology, 2008

MicroRNAs (miRNAs) are non-coding RNAs that bind to target mRNA leading to translational arrest o... more MicroRNAs (miRNAs) are non-coding RNAs that bind to target mRNA leading to translational arrest or mRNA degradation. To study miRNA-mediated regulation of osteogenesis and chondrogenesis, we compared the expression of 35 miRNAs in osteoblasts and chondroblasts derived from mouse marrow stromal cells (MSCs). Differentiation of MSCs resulted in up-or downregulation of several miRNAs, with miR-199a expression being over 10-fold higher in chondroblasts than in undifferentiated MSCs. In addition, miR-124a was strongly upregulated during chondrogenesis while the expression of miR-96 was substantially suppressed. A systems biological analysis of the potential miRNA target genes and their interaction networks was combined with promoter analysis. These studies link the differentially expressed miRNAs to collagen synthesis and hypoxia, key pathways related to bone and cartilage physiology. The global regulatory networks described here suggest for the fi rst time how miRNAs and transcription factors are capable of fi ne-tuning the osteogenic and chondrogenic differentiation of mouse MSCs.

Journal of Biomechanical Science and Engineering, 2007

Osteocyte is the most abundant cell type in bone, and the only cell type located inside the miner... more Osteocyte is the most abundant cell type in bone, and the only cell type located inside the mineralized matrix. The striking structural design of bone predicts an important role for osteocytes in determining bone structure and function. Osteocytes are connected with each other via gap junctions and form a three dimensional cellular network in mineralized bone matrix. Recently, it has been shown that osteocytes are not only passive bystanders, but also have an active regulatory role in whole body phosphate and calcium metabolism. Osteocytes are cells which sense mechanical loading in bone. They respond to mechanical stimuli by producing and secreting several bioactive substances including nitric oxide and prostaglandins and thus transmit messages of loading to effector cells, like osteoblasts and osteoclasts. Present data suggest that osteocytes actively inhibit osteoclastic bone resorption. Whenever osteocytes die, this inhibitory effect is turned off and osteoclasts are activated. This mechanism of action could explain targeted remodeling in the region of stress induced microcracks happened. Osteocytes can also modulate osteoblasts function. In conclusion, osteocytes sense mechanical stimuli, transmit signals through cellular network and regulate osteoblast and osteoclast function during bone remodeling.

Journal of Histochemistry and Cytochemistry, Oct 1, 1990

Bone resorbing osteodasts form a specific microfilament structure at the attachment area, in whic... more Bone resorbing osteodasts form a specific microfilament structure at the attachment area, in which vinculin and tabin appear as a doubbe-cirde structure and F-actin fills the space between these cirdes. This distribution of microfilaments is associated with the resorption lacunae, and F-actin, yinculin, and talin zones correspond roughly to the edges of the lacunae. In the present work, we examined by immunofluorescence the effects of calcitonin (CT) and prostaglandin E2 (PGE4, inhibitors of osteodastic activity, as well as dibutyryl cyclic AMP (Bt2cAMP) and cytochabasin B, on the microfilament organization in resorbing osteoclasts. CT, PGE2, and Bt2cAMP rapidly dispersed the specific microfilament structure in resorbing osteoclasts. All microfil-I Supported by a grant from Sigrid Jus#{233}lius Foundation and Paulo Foundation.

Journal of Cell Biology, Nov 15, 1991

During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption ... more During bone resorption, osteoclasts form a tight attachment, the sealing zone, around resorption lacunae. Vitronectin receptor has previously been shown to be expressed in osteoclasts and it has been suggested that it mediates the tight attachment at the sealing zone. In this study we have shown that glycinearginine-glycine-aspartic acid-serine pentapeptide inhibits bone resorption by isolated osteoclasts and drastically changes the morphology of the osteoclasts. When the vitronectin receptor was localized by immunofluorescence in rat and chicken osteoclasts cultured on bone slices, it was found to be distributed throughout the osteoclast cell membrane except in the sealing zone areas. Immunoperoxidase staining of rat bone sections at the light microscopical level also revealed in-0 TEOC1.ASTs are multinucleated cells responsible for

12th European Congress of Endocrinology, Apr 1, 2010

Journal of Biomechanical Science and Engineering, 2007

Osteocyte is the most abundant cell type in bone, and the only cell type located inside the miner... more Osteocyte is the most abundant cell type in bone, and the only cell type located inside the mineralized matrix. The striking structural design of bone predicts an important role for osteocytes in determining bone structure and function. Osteocytes are connected with each other via gap junctions and form a three dimensional cellular network in mineralized bone matrix. Recently, it has been shown that osteocytes are not only passive bystanders, but also have an active regulatory role in whole body phosphate and calcium metabolism. Osteocytes are cells which sense mechanical loading in bone. They respond to mechanical stimuli by producing and secreting several bioactive substances including nitric oxide and prostaglandins and thus transmit messages of loading to effector cells, like osteoblasts and osteoclasts. Present data suggest that osteocytes actively inhibit osteoclastic bone resorption. Whenever osteocytes die, this inhibitory effect is turned off and osteoclasts are activated. This mechanism of action could explain targeted remodeling in the region of stress induced microcracks happened. Osteocytes can also modulate osteoblasts function. In conclusion, osteocytes sense mechanical stimuli, transmit signals through cellular network and regulate osteoblast and osteoclast function during bone remodeling.

BoneKEy Reports, 2013

We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytoti... more We analyzed the characteristics of degraded bone matrix-delivering vesicles along the transcytotic route from the ruffled border to the functional secretory domain (FSD) in bone-penetrating osteoclasts. Cells of rat or human origin were cultured on bovine bone slices and analyzed via confocal microscopy. Helix pomatia lectin binding indicated that transcytotic vesicles expose aberrant N-acetylgalactosamine glycoconjugates, which is associated with a poor prognosis for a range of metastasizing human adenocarcinomas. Transcytotic vesicles fuse with the autophagosomal compartments and represent raft concentrates. Furthermore, the results of a vertical vesicle analysis suggest that multiple vesicle populations arise from the ruffled border and that some of these vesicles undergo a maturation process along the transcytotic route. Finally, our data suggest that the targeting of these membrane pathways may be determined by a novel F-actin-containing and FSD-circumscribing molecular barrier.

The Journal of cell biology, 1990

Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts ... more Microsomal membrane vesicles prepared either from chicken medullary bone or isolated osteoclasts were shown to have ATP-dependent H(+)-transport activity. This activity was N-ethylmaleimide-sensitive but resistant to oligomycin and orthovanadate, suggesting a vacuolar-type ATPase. Furthermore, immunological cross-reactivity of 60- and 70-kD osteoclast membrane antigens with Neurospora crassa vacuolar ATPase was observed when analyzed by immunoblotting. Same antibodies labeled only osteoclasts in chicken and rat bone in immunohistochemistry. Immunoelectronmicroscopy localized these antigens in apical membranes of rat osteoclasts and kidney intercalated cells of inner stripe of outer medulla. Pretreatment of animals with parathyroid hormone enhanced the immunoreaction in the apical membranes of osteoclasts. No immunoreaction was seen in osteoclasts when antibodies against gastric H+,K(+)-ATPase were used. These results suggest that osteoclast resorbs bone by secreting protons through ...

Journal of Histochemistry & Cytochemistry, 1989

The appearance of carbonic anhydrase isoenzymes in rat liver and skeletal muscle during fetal and... more The appearance of carbonic anhydrase isoenzymes in rat liver and skeletal muscle during fetal and postnatal development was studied with immunohistochemistry. In the 12-day fetus the early strong expression of CA I in hepatocytes was partially replaced by the expression of CA II and CA III during late prenatal development. In the 20-day fetus the staining intensity of CA III was equal to that of a mature female rat. In the male, the staining intensity in hepatocytes clearly increased during sexual maturation. Immunoelectron microscopy showed diffuse cytoplasmic and nucleoplasmic staining of CA III in hepatocytes. The time-dependent expression of the isoenzymes in hepatocytes may reflect a different metabolic function of these structurally closely related isoenzymes. In skeletal muscle, CA III was the only isoenzyme detected during development. In late prenatal and early postnatal stages all muscle fibers contained about equal amounts of CA III. The differentiation of the muscle acco...

Journal of Bone and Mineral Research, 2006

Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover ... more Osteocytes are suggested to have a crucial role in the initial resorptive phase of bone turnover after microdamage. To study the role of osteocytes in targeted remodeling, we developed an in vitro model, in which osteocytes can be locally damaged and their interactions with bone marrow cells studied. Our results show that the damaged osteocytes activate the osteoclast precursors by soluble factors and thus can control the initial phase of targeted remodeling. Introduction: Microdamage in bone contributes to fractures and acts as a stimulus for bone remodeling. Besides the targeted remodeling, some remodeling may also be random to serve metabolic purposes. Osteocytes have been considered to provide a crucial role in the activation of osteoclastic bone resorption adjacent to the damaged site. This study was aimed to develop a relevant in vitro model of the targeted remodeling and to show that damaged osteocytes can induce the initial bone resorptive stage. Materials and Methods: We developed a new device, in which osteocyte-like cell line MLO-Y4 cells were 3D cultured, subjected to local scratching, and assayed for cell viability. NIH3T3-3 cells were used as a control. Bone marrow cells were cultured on the top of the mechanically damaged MLO-Y4 cells, and the formation of TRACP + cells was assayed. Additionally, the conditioned medium from scratched cultures was added to bone marrow cultures, and the TRACP activity in cell lysates was quantified. The macrophage-colony stimulating factor (M-CSF) and RANKL secretion in the conditioned medium was assayed by ELISA. Results: Scratching induced the death of MLO-Y4 cells. When bone marrow cells were cultured over the gel-embedded MLO-Y4 cells, the application of mechanical scratching induced TRACP + cell differentiation on gel surface. The cells with TRACP + could be observed in the very restricted region along the scratching path. Additionally, mechanically damaged osteocytes secreted M-CSF and RANKL, and the conditioned medium showed the potential to induce TRACP + cells in bone marrow culture. Conclusions: These findings indicate that soluble factors secreted from damaged osteocytes can locally induce and activate the initial phase of osteoclastic cell formation. This study directly shows the association between the damaged osteocytes and the initiation of resorptive stage in bone remodeling.

Biochemical and Biophysical Research Communications, 2000

The Carbonic Anhydrase II (CAII) gene that encodes an enzyme involved in proton production is exp... more The Carbonic Anhydrase II (CAII) gene that encodes an enzyme involved in proton production is expressed in several cell types including monocyte/macrophagederived osteoclasts. We have analyzed the regulation of the chicken CAII promoter/reporter construct by nuclear hormone receptors of the VDR subfamily in HD11 avian macrophages. The CAII expression is stimulated by 1,25-dihydroxyvitamin D 3 but not by 9-cis retinoic acid and repressed by VDR overexpression due to RXR squelching. It is also stimulated by alltrans retinoic acid only when RAR␣ is overexpressed, and is dependent on a RARE located in the distal part of the promoter and bound by RAR␣ homodimer. Finally, in macrophages, unlike in erythrocytes, the CAII promoter is unresponsive to thyroid hormone. Our results demonstrate the first retinoic acid response element in the CAII promoter and show that according to cell type, different nuclear receptors of the VDR subfamily can regulate the CAII gene.

PLOS ONE, May 3, 2013

Objective: Osteocalcin (OC), a bone-derived protein, has been implicated in the regulation of glu... more Objective: Osteocalcin (OC), a bone-derived protein, has been implicated in the regulation of glucose and energy metabolism. Young adults born with very low birth weight (VLBW) have altered glucose regulation and lower bone mineral density (BMD) compared with those born at term. The aim of this study was to explore the association between bone and glucose metabolism in healthy young adults born prematurely or at term. Methods: The cohort of this cross-sectional study comprised 332 non-diabetic young adults (age 18 to 27 years) born either preterm with VLBW (n = 163) or at term (n = 169). OC, carboxylated osteocalcin (cOC) and markers of glucose metabolism were measured at fasting and after a 75-g oral glucose tolerance test (OGTT). Results: VLBW adults were shorter, had lower BMD (p,0.001) and higher fasting OC (p = 0.027) and cOC (p = 0.005) than term-born subjects. They also had higher 2-hour insulin (p = 0.001) and glucose (p = 0.037) concentrations. OGTT induced a significant reduction in OC (p,0.001), similar in both groups. OC reduction was not associated with OGTT-induced increases in insulin (p = 0.54). However, fasting total OC and cOC correlated negatively with fasting insulin after adjustment for age, gender, BMD and VLBW status (r = 20.182, p = 0.009 and r = 20.283, p,0.001, respectively). Conclusion: Adults born with VLBW have higher OC and cOC than their peers born at term. This may in part reflect the mechanisms that underlie their lower BMD and decreased insulin sensitivity. Serum OC appears to be negatively associated with long-term glucose regulation whereas acute changes during OGTT may be mediated via other mechanisms.

American Journal of Obstetrics and Gynecology, 2002

Objective: Estrogen replacement therapy after menopause reduces the incidence of arterial disease... more Objective: Estrogen replacement therapy after menopause reduces the incidence of arterial disease and cerebrovascular events. The reduced incidence also seems to be due to a positive effect of estrogens on brain blood flow as shown by a decrease in the carotid ...