Biomedical subjects

Thorsten Kirsch

Publications and source records attributed to Thorsten Kirsch.

12 recordsLinked to original sources

PubMed2006

Annexin V/beta5 integrin interactions regulate apoptosis of growth plate chondrocytes.

Apoptosis of terminally differentiated chondrocytes allows the replacement of growth plate cartilage by bone. Despite its importance, little is known about the regulation of chondrocyte apoptosis. We show that overexpression of annexin V, which binds to the cytoplasmic domain of beta5 integrin and protein kinase C alpha (PKCalpha), stimulates apoptotic events in hypertrophic growth plate chondrocytes. To determine whether the balance between the interactions of annexin V/beta5 integrin and annexin V/active PKCalpha play a role in the regulation of terminally differentiated growth plate chondrocyte apoptosis, a peptide mimic of annexin V (Penetratin (Pen)-VVISYSMPD) that binds to beta5 integrin but not to PKCalpha was used. This peptide stimulated apoptotic events in growth plate chondrocytes. Suppression of annexin V expression using small interfering ribonucleic acid decreased caspase-3 activity and increased cell viability in Pen-VVISYSMPD-treated growth plate chondrocytes. An activator of PKC resulted in a further decrease of cell viability and further increase of caspase-3 activity in Pen-VVISYSMPD-treated growth plate chondrocytes, whereas inhibitors of PKCalpha led to an increase of cell viability and decrease of caspase-3 activity of Pen-VVISYSMPD-treated cells. These findings suggest that binding of annexin V to active PKCalpha stimulates apoptotic events in growth plate chondrocytes and that binding of annexin Vto beta5 integrin controls these interactions and ultimately apoptosis.

Animals↗

PubMed2006

Determinants of pathological mineralization.

PURPOSE OF REVIEW: Physiological mineralization is necessary for the formation of skeletal tissues and for their appropriate functions during adulthood. Pathological or ectopic mineralization of soft tissues, including articular cartilage and cardiovascular tissues, leads to morbidity and mortality. Recent findings suggest that the mechanisms and factors regulating physiological mineralization may be identical or similar to those regulating ectopic mineralization. Therefore, the purpose of this review is to describe the current knowledge of mechanisms and determinants that regulate physiological mineralization and how these determinants can be used to understand ectopic mineralization better. RECENT FINDINGS: Recent findings have indicated that physiological and pathological mineralization are initiated by matrix vesicles, membrane-enclosed particles released from the plasma membrane of mineralization-competent cells. An understanding of how these vesicles initiate the physiological mineralization process may provide novel therapeutic strategies to prevent ectopic mineralization. In addition, other regulators (activators and inhibitors) of physiological mineralization have been identified and characterized, and evidence indicates that the same factors also contribute to the regulation of ectopic mineralization. Finally, programmed cell death (apoptosis) may be a contributor to physiological mineralization, and if occurring after tissue injury may induce ectopic mineralization and mineralization-related differentiation events in the injured area and surrounding areas. SUMMARY: This review describes how the understanding of mechanisms and factors regulating physiological mineralization can be used to develop new therapeutic strategies to prevent pathological or ectopic mineralization events.

Apoptosis↗

PubMed2005

Annexin V and terminal differentiation of growth plate chondrocytes.

Terminal differentiation and mineralization are the final events in endochondral bone formation and allow the replacement of cartilage by bone. Retinoic acid (RA) stimulates these events, including upregulation of expression and activity of alkaline phosphatase (APase), expression of annexins II, V, and VI proteins, which bind to membranes and form Ca(2+) channels, expression of osteocalcin and runx2, another mineralization-related protein and terminal differentiation-related transcription factor, and ultimately mineralization. Chelating cytosolic Ca(2+) with BAPTA-AM, interfering with annexin Ca(2+) channel activities using K-201, a specific annexin Ca(2+) channel blocker, or suppression of annexin V expression using siRNA inhibited these events. Overexpression of annexin V in embryonic chicken growth plate chondrocytes resulted in an increase of cytoplasmic Ca(2+) concentration, [Ca(2+)](i) similar to [Ca(2+)](i) increase in RA-treated cultures. Overexpression of annexin V also resulted in upregulation of annexin II, annexin VI, osteocalcin, and runx2 gene expression, expression and activity of APase, and ultimately stimulation of mineralization. K-201 inhibited upregulation of osteocalcin and runx2 gene expression, APase expression and activity, and mineralization in annexin V-overexpressing growth plate chondrocytes. These findings indicate that annexins II, V, and VI alter Ca(2+) homeostasis in growth plate chondrocytes thereby regulating terminal differentiation and mineralization events. Overexpression of annexin V is sufficient to stimulate these terminal differentiation events in growth plate chondrocytes, whereas suppression of annexin V expression inhibits these events.

Alkaline Phosphatase↗

PubMed2005

The conserved core domains of annexins A1, A2, A5, and B12 can be divided into two groups with different Ca2+-dependent membrane-binding properties.

The hallmark of the annexin super family of proteins is Ca(2+)-dependent binding to phospholipid bilayers, a property that resides in the conserved core domain of these proteins. Despite the structural similarity between the core domains, studies reported herein showed that annexins A1, A2, A5, and B12 could be divided into two groups with distinctively different Ca(2+)-dependent membrane-binding properties. The division correlates with the ability of the annexins to form Ca(2+)-dependent membrane-bound trimers. Site-directed spin-labeling and Forster resonance energy transfer experimental approaches confirmed the well-known ability of annexins A5 and B12 to form trimers, but neither method detected self-association of annexin A1 or A2 on bilayers. Studies of chimeras in which the N-terminal and core domains of annexins A2 and A5 were swapped showed that trimer formation was mediated by the core domain. The trimer-forming annexin A5 and B12 group had the following Ca(2+)-dependent membrane-binding properties: (1) high Ca(2+) stoichiometry for membrane binding ( approximately 12 mol of Ca(2+)/mol of protein); (2) binding to membranes was very exothermic (> -60 kcal/ mol of protein); and (3) binding to bilayers that were in the liquid-crystal phase but not to bilayers in the gel phase. In contrast, the nontrimer-forming annexin A1 and A2 group had the following Ca(2+)-dependent membrane-binding properties: (1) lower Ca(2+) stoichiometry for membrane binding (<or=4 mol of Ca(2+)/mol of protein); (2) binding to membranes was relatively less exothermic (< -33 kcal/ mol of protein); and (3) binding to bilayers that were in either the liquid-crystal phase or gel phase. The biological implications of this subdivision are discussed.

Amino Acid Sequence↗

PubMed2005

Annexins - their role in cartilage mineralization.

Annexins II, V and VI are highly expressed by hypertrophic and terminally differentiated growth plate chondrocytes and by osteoblasts. Because of the localization of annexins in areas of cartilage and bone mineralization, we hypothesized that these annexins play a regulatory role in the mineralization process. In this article we review the function of annexins II, V and VI in physiological mineralization of skeletal tissues and in pathological mineralization of articular cartilage.

Animals↗

PubMed2005

Syndecan-3: a cell-surface heparan sulfate proteoglycan important for chondrocyte proliferation and function during limb skeletogenesis.

Syndecans are single-pass integral membrane components that serve as co-receptors for growth factors and cytokines and can elicit signal transduction via their cytoplasmic tails. We review here previous studies from our groups on syndecan-3 biology and function in the growth plates of developing long bones in chick and mouse embryos. Gain- and loss-of-function data indicate that syndecan-3 has important roles in restricting mitotic activity to the proliferative zone of growth plate and may do so in close cooperation and interaction with the signaling molecule Indian hedgehog (IHH). Biochemical and protein-modeling data suggest a dimeric/oligomeric syndecan-3 configuration on the chondrocyte's cell surface. Analyses of embryos misexpressing syndecan-3 or lacking IHH provide further clues on syndecan-3/IHH interdependence and interrelationships. The data and the conclusions reached provide insights into mechanisms fine-tuning chondrocyte proliferation, maturation, and function in the developing and growing skeleton and into how abnormalities in these fundamental mechanisms may subtend human congenital pathologies, including osteochondromas in hereditary multiple exostoses syndrome.

Animals↗

PubMed2005

Role of the progressive ankylosis gene (ank) in cartilage mineralization.

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PP(i)) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PP(i) concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PP(i) concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PP(i) levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (P(i)) and gene expression of the type III Na(+)/P(i) cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PP(i) transport to the extracellular milieu, initial hydrolysis of PP(i) to P(i), P(i)-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PP(i), and subsequent mineralization.

Alkaline Phosphatase↗

PubMed2004

Effect of COX-2-specific inhibition on fracture-healing in the rat femur.

BACKGROUND: Nonsteroidal anti-inflammatory medications have been shown to delay fracture-healing. COX-2-specific inhibitors such as celecoxib have recently been approved for human use. Our goal was to determine, mechanically, histologically, morphologically, and radiographically, whether COX-2-specific inhibition affects bone-healing. METHODS: A nondisplaced unilateral fracture was created in the right femur of fifty-seven adult male rats. Rats were given no drug, indomethacin (1 mg/kg/day), or celecoxib (3 mg/kg/day) daily, starting on postoperative day 1. Fractures were analyzed at four, eight, and twelve weeks after creation of the fracture. Callus and bridging bone formation was assessed radiographically. The amounts of fibrous tissue, cartilage, woven bone, and mature bone formation were determined histologically. Morphological changes were assessed to determine fibrous healing, callus formation, and bone-remodeling. Callus strength and stiffness were assessed biomechanically with three-point bending tests. RESULTS: At four weeks, only the indomethacin group showed biomechanical and radiographic evidence of delayed healing. Although femora from rats treated with celecoxib appeared to have more fibrous tissue than those from untreated rats at four and eight weeks, radiographic signs of callus formation, mechanical strength, and stiffness did not differ significantly between the groups. By twelve weeks, there were no significant differences among the three groups. CONCLUSIONS: Postoperative administration of celecoxib, a COX-2-specific inhibitor, did not delay healing as seen at twelve weeks following fracture in adult rat femora. At four and eight weeks, fibrous healing predominated in the celecoxib group as compared with the findings in the untreated group; however, mechanical strength and radiographic signs of healing were not significantly inhibited. CLINICAL RELEVANCE: Many orthopaedists rely on narcotic analgesia for postfracture and postoperative pain, despite deleterious side effects and morbidity. Traditional nonsteroidal anti-inflammatory medications have been shown to delay fracture union. This effect may be smaller with COX-2-specific inhibitors.

Animals↗

PubMed2003

Functional differences between growth plate apoptotic bodies and matrix vesicles.

UNLABELLED: Mineralization often occurs in areas of apoptotic changes. Our findings indicate that physiological mineralization is mediated by matrix vesicles. These matrix vesicles use mechanisms to induce mineralization that are different from the mechanisms used by apoptotic bodies released from apoptotic cells. Therefore, different therapeutic approaches must be chosen to inhibit pathological mineralization depending on the mechanism of mineralization (matrix vesicles versus apoptotic bodies). INTRODUCTION: Physiological mineralization in growth plate cartilage is restricted to regions of terminally differentiated and apoptotic chondrocytes. Pathological mineralization of tissues also often occurs in areas of apoptosis. We addressed the question of whether apoptotic changes control mineralization events or whether both events are regulated independently. METHODS: To induce mineralization, we treated growth plate chondrocytes with retinoic acid (RA); apoptosis in these cells was induced by treatment with staurosporine, anti-Fas, or TNFalpha. The degrees of mineralization and apoptosis were determined, and the structure and function of matrix vesicles and apoptotic bodies were compared. RESULTS: Release of matrix vesicles and mineralization in vivo in the growth plate occurs earlier than do apoptotic changes. To determine the functional relationship between apoptotic bodies and matrix vesicles, growth plate chondrocytes were treated with RA to induce matrix vesicle release and with staurosporine to induce release of apoptotic bodies. After 3 days, approximately 90% of staurosporine-treated chondrocytes were apoptotic, whereas only 2-4% of RA-treated cells showed apoptotic changes. RA- and staurosporine-treated chondrocyte cultures were mineralized after 3 days. Matrix vesicles isolated from RA-treated cultures and apoptotic bodies isolated from staurosporine-treated cultures were associated with calcium and phosphate. However, matrix vesicles were bigger than apoptotic bodies. Furthermore, matrix vesicles but not apoptotic bodies contained alkaline phosphatase and Ca2+ channel-forming annexins II, V, and VI. Consequently, matrix vesicles but not apoptotic bodies were able to take up Ca2+ and form the first mineral phase inside their lumen. Mineralization of RA-treated cultures was inhibited by antibodies specific for annexin V but not mineralization of staurosporine-treated cultures. CONCLUSION: Physiological mineralization of growth plate chondrocytes is initiated by specialized matrix vesicles and requires alkaline phosphatase and annexins. In contrast, mineral formation mediated by apoptotic bodies occurs by a default mechanism and does not require alkaline phosphatase and annexins.

Alkaline Phosphatase↗

PubMed2002

Annexin-mediated Ca2+ influx regulates growth plate chondrocyte maturation and apoptosis.

Maturation of epiphyseal growth plate chondrocytes plays an important role in endochondral bone formation. Previously, we demonstrated that retinoic acid (RA) treatment stimulated annexin-mediated Ca(2+) influx into growth plate chondrocytes leading to a significant increase in cytosolic Ca(2+), whereas K-201, a specific annexin Ca(2+) channel blocker, inhibited this increase markedly. The present study addressed the hypothesis that annexin-mediated Ca(2+) influx into growth plate chondrocytes is a major regulator of terminal differentiation, mineralization, and apoptosis of these cells. We found that K-201 significantly reduced up-regulation of expression of terminal differentiation marker genes, such as cbfa1, alkaline phosphatase (APase), osteocalcin, and type I collagen in RA-treated cultures. Furthermore, K-201 inhibited up-regulation of annexin II, V, and VI gene expression in these cells. RA-treated chondrocytes released mineralization-competent matrix vesicles, which contained significantly higher amounts of annexins II, V, and VI as well as APase activity than vesicles isolated from untreated or RA/K-201-treated cultures. Consistently, only RA-treated cultures showed significant mineralization. RA treatment stimulated the whole sequence of terminal differentiation events, including apoptosis as the final event. After a 6-day treatment gene expression of bcl-2, an anti-apoptotic protein, was down-regulated, whereas caspase-3 activity and the percentage of TUNEL-positive cells were significantly increased in RA-treated cultures compared with untreated cultures. Interestingly, the cytosolic calcium chelator BAPTA-AM and K-201 protected RA-treated chondrocytes from undergoing apoptotic changes, as indicated by higher bcl-2 gene expression, reduced caspase-3 activity, and the percentage of TUNEL-positive cells. In conclusion, annexin-mediated Ca(2+) influx into growth plate chondrocytes is a positive regulator of terminal differentiation, mineralization, and apoptosis events in growth plate chondrocytes.

Animals↗

PubMed2002

Syndecan-3 is a selective regulator of chondrocyte proliferation.

Chondrocyte proliferation is important for skeletal development and growth, but the mechanisms regulating it are not completely clear. Previously, we showed that syndecan-3, a cell surface heparan sulfate proteoglycan, is expressed by proliferating chondrocytes in vivo and that proliferation of cultured chondrocytes in vitro is sensitive to heparitinase treatment. To further establish the link between syndecan-3 and chondrocyte proliferation, additional studies were carried out in vivo and in vitro. We found that the topographical location of proliferating chondrocytes in developing chick long bones changes with increasing embryonic age and that syndecan-3 gene expression changes in a comparable manner. For in vitro analysis, mitotically quiescent chondrocytes were exposed to increasing amounts of fibroblast growth factor-2 (FGF-2). Proliferation was stimulated by as much as 8-10-fold within 24 h; strikingly, this stimulation was significantly prevented when the cells were treated with both fibroblast growth factor-2 (FGF-2) and antibodies against syndecan-3 core protein. This neutralizing effect was dose-dependent and elicited a maximum of 50-60% inhibition. To establish specificity of neutralizing effect, cultured chondrocytes were exposed to FGF-2, insulin-like growth factor-1, or parathyroid hormone, all known mitogens for chondrocytes. The syndecan-3 antibodies interfered only with FGF-2 mitogenic action, but not that of insulin-like growth factor-1 or parathyroid hormone. Protein cross-linking experiments indicated that syndecan-3 is present in monomeric, dimeric, and oligomeric forms on the chondrocyte surface. In addition, molecular modeling indicated that contiguous syndecan-3 molecules might form stable complexes by parallel pairing of beta-sheet segments within the ectodomain of the core protein. In conclusion, the results suggest that syndecan-3 is a direct and selective regulator of the mitotic behavior of chondrocytes and its role may involve formation of dimeric/oligomeric structures on their cell surface.

Animals↗

PubMed2002

Retinoic acid stimulates annexin-mediated growth plate chondrocyte mineralization.

Biomineralization is a highly regulated process that plays a major role during the development of skeletal tissues. Despite its obvious importance, little is known about its regulation. Previously, it has been demonstrated that retinoic acid (RA) stimulates terminal differentiation and mineralization of growth plate chondrocytes (Iwamoto, M., I.M. Shapiro, K. Yagumi, A.L. Boskey, P.S. Leboy, S.L. Adams, and M. Pacifici. 1993. Exp. Cell Res. 207:413-420). In this study, we provide evidence that RA treatment of growth plate chondrocytes caused a series of events eventually leading to mineralization of these cultures: increase in cytosolic calcium concentration, followed by up-regulation of annexin II, V, and VI gene expression, and release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles. Cotreatment of growth plate chondrocytes with RA and BAPTA-AM, a cell permeable Ca2+ chelator, inhibited the up-regulation of annexin gene expression and mineralization of these cultures. Interestingly, only matrix vesicles isolated from RA-treated cells that contained annexins, were able to take up Ca2+ and mineralize, whereas vesicles isolated from untreated or RA/BAPTA-treated cells, that contained no or only little annexins were not able to take up Ca2+ and mineralize. Cotreatment of chondrocytes with RA and EDTA revealed that increases in the cytosolic calcium concentration were due to influx of extracellular calcium. Interestingly, the novel 1,4-benzothiazepine derivative K-201, a specific annexin Ca2+ channel blocker, or antibodies specific for annexin II, V, or VI inhibited the increases in cytosolic calcium concentration in RA-treated chondrocytes. These findings indicate that annexins II, V, and VI form Ca2+ channels in the plasma membrane of terminally differentiated growth plate chondrocytes and mediate Ca2+ influx into these cells. The resulting increased cytosolic calcium concentration leads to a further up-regulation of annexin II, V, and VI gene expression, the release of annexin II-, V-, VI- and alkaline phosphatase-containing matrix vesicles, and the initiation of mineralization by these vesicles.

Alkaline Phosphatase↗