Methods and insights from the characterization of osteoprogenitor cells of bats (Mammalia: Chiroptera).

Osteoprogenitor cells contribute to the development and maintenance of skeletal tissues. Bats are unique model taxa whose cellular processes are poorly understood, especially in regards to skeletal biology. Forelimb bones of bats, unlike those of terrestrial mammals, bend during flight and function in controlled deformation. As a first step towards understanding the molecular processes governing deposition of this flexible bone matrix, we provide the first method for isolation and differentiation of cell populations derived from the bone marrow and cortical bone of bats, and compare results with those harvested from C57BL/6J mice. Osteogenic capacity of these cells was assessed via absolute quantitative real-time PCR (qPCR) and through quantification of in vitro mineral deposition. Results indicate the differentiated bone cells of bats display significantly lower gene expression of known osteogenic markers (Runt-related transcription factor (RUNX2), osteocalcin (BGLAP) and osterix (SP7)), and deposit a less-mineralized matrix compared with murine controls. By characterizing the in vitro performance of osteoprogenitor cells throughout differentiation and matrix production, this study lays the ground work for in vitro manipulations of bat stem and osteoprogenitor cells and extends our understanding of the cellular diversity across mammals that occupy different habitats.

Comprehensive characterization of mesenchymal stem cells from human placenta and fetal membrane and their response to osteoactivin stimulation.

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy and can be isolated from various sources of human adult tissues such as bone marrow (BM-MSC) and adipose tissue. However, cells from these tissues must be obtained through invasive procedures. We, therefore, characterized MSCs isolated from fresh placenta (Pl-MSC) and fetal membrane (Mb-MSC) through morphological and fluorescent-activated cell sorting (FACS). MSC frequency is higher in membrane than placenta (2.14% ± 0.65 versus 15.67% ± 0.29%). Pl/Mb-MSCs in vitro expansion potential was significantly higher than BM-MSCs. We demonstrated that one of the MSC-specific marker is sufficient for MSC isolation and that culture in specific media is the optimal way for selecting very homogenous MSC population. These MSCs could be differentiated into mesodermal cells expressing cell markers and cytologic staining consistent with mature osteoblasts and adipocytes. Transcriptomic analysis and cytokine arrays demonstrated broad similarity between placenta- and membrane-derived MSCs and only discrete differences with BM-MSCs with enrichment of networks involved in bone differentiation. Pl/Mb-MSCs displayed higher osteogenic differentiation potential than BM-MSC when their response to osteoactivin was evaluated. Fetal-tissue-derived mesenchymal cells may, therefore, be considered as a major source of MSCs to reach clinical scale banking in particular for bone regeneration.

Exploratory study on the effect of osteoactivin on bone formation in the rat critical-size calvarial defect model.

BACKGROUND AND OBJECTIVE: Osteoactivin is a novel glycoprotein shown to exhibit an important role in regulating osteoblast differentiation and function. The aim of the present study was to evaluate the potential of osteoactivin to support bone regeneration using an established defect model. MATERIAL AND METHODS: Critical-size, 8-mm-diameter through-and-through calvarial osteotomy defects were created in 60 adult male Sprague-Dawley rats. Test animals received 0.1 mL of osteoactivin in phosphate-buffered saline (50 µg/mL) soak-loaded onto an absorbable collagen sponge. Controls received 0.1 mL of phosphate-buffered saline soak-loaded onto the absorbable collagen sponge or no further intervention (sham-surgery). The animals were euthanized 2 and 4 wk after treatment and histometric analyses were performed. RESULTS: The absorbable collagen sponge control (mean ± standard deviation: 40.9 ± 26.9%) showed borderline significant greater bone fill compared with sham-surgery (22.9 ± 15.8%; p = 0.10) and osteoactivin (20.2 ± 11.8%; p = 0.07) treatments at 2 wk. In contrast, osteoactivin (84.7 ± 15.8%) showed significantly greater bone fill than sham-surgery (28.4 ± 9.6%; p < 0.001) and absorbable collagen sponge (41.8 ± 22.1%; p < 0.001) at 4 wk. No animals receiving sham-surgery or absorbable collagen sponge exhibited complete bone fill at 4 wk while 70% of the animals receiving osteoactivin showed complete bone fill. CONCLUSION: Osteoactivin demonstrates a significant potential to support bone regeneration/formation. Studies using discriminating large animal models are necessary to explore clinical application for periodontal and craniofacial indications.

Different effects on bone strength and cell differentiation in pre pubertal caloric restriction versus hypothalamic suppression.

Hypothalamic amenorrhea and energy restriction during puberty affect peak bone mass accrual. One hypothesis suggests energy restriction alters hypothalamic function resulting in suppressed estradiol levels leading to bone loss. However, both positive and negative results have been reported regarding energy restriction and bone strength. Therefore, the purpose of this study was to investigate energy restriction and hypothalamic suppression during pubertal onset on bone mechanical strength and the osteogenic capacity of bone marrow-derived cells in two models: female rats treated with gonadotropin releasing hormone antagonists (GnRH-a) or 30% energy restriction. At 23 days of age, female Sprague Dawley rats were assigned to three groups: control group (C, n=10), GnRH-a group (n=10), and Energy Restriction (ER, n=12) group. GnRH-a animals received daily injections for 27 days. The animals in the ER group received 70% of the control animals' intake. After sacrifice (50 days of age), body weight, uterine and muscle weights were measured. Bone marrow-derived stromal cells were cultured and assayed for proliferation and differentiation into osteoblasts. Outcome measures included bone strength, bone histomorphometry and architecture, serum IGF-1 and osteocalcin. GnRH-a suppressed uterine weight, decreased osteoblast proliferation, bone strength, trabecular bone volume and architecture compared to control. Elevated serum IGF-1 and osteocalcin levels and body weight were found. The ER model had an increase in osteoblast proliferation compared to the GnRH-a group, similar bone strength relative to body weight and increased trabecular bone volume in the lumbar spine compared to control. The ER animals were smaller but had developed bone strength sufficient for their size. In contrast, suppressed estradiol via hypothalamic suppression resulted in bone strength deficits and trabecular bone volume loss. In summary, our results support the hypothesis that during periods of nutritional stress the increased vertebral bone volume may be an adaptive mechanism to store mineral which differs from suppressed estradiol resulting from hypothalamic suppression.

Src is a major signaling component for CTGF induction by TGF-beta1 in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta1 (TGF-beta1) where it acts as a downstream mediator of TGF-beta1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk, and Smad signaling for CTGF induction by TGF-beta1 in osteoblasts; however, the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-beta1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-beta1. Additionally, inhibiting Src activation prevented Erk activation, Smads 2 and 3 activation and nuclear translocation by TGF-beta1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway directly by mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059, it inhibited TGF-beta1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) of the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. These data demonstrate that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-beta1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts.

Molecular requirements for induction of CTGF expression by TGF-beta1 in primary osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine rich, extracellular matrix protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. In osteoblasts, CTGF is induced by TGF-beta1 where it acts as a downstream mediator of TGF-beta1 induced matrix production. The molecular mechanisms that control CTGF induction by TGF-beta1 in osteoblasts are not known. To assess the role of individual Smads in mediating the induction of CTGF by TGF-beta1, we used specific Smad siRNAs to block Smad expression. These studies demonstrated that Smads 3 and 4, but not Smad 2, are required for TGF-beta1 induced CTGF promoter activity and expression in osteoblasts. Since the activation of MAPKs (Erk, Jnk and p38) by TGF-beta1 is cell type specific, we were interested in determining the role of individual MAPKs in TGF-beta1 induction of CTGF promoter activity and expression. Using dominant negative (DN) mutants for Erk, Jnk and p38, we demonstrated that the expression of DN-Erk caused a significant inhibition of TGF-beta1 induced CTGF promoter activity. In contrast, the expression of DN-p38 or DN-Jnk failed to inhibit activation of CTGF promoter activity. To confirm the vital role of Erk, we used the Erk inhibitor (PD98059) to block its activation, demonstrating that it prevented TGF-beta1 activation of the CTGF promoter and up-regulation of CTGF expression in osteoblasts. Since Src can also act as a downstream signaling effector for TGF-beta in some cell types, we determined its role in TGF-beta1 induction of CTGF in osteoblasts. Treatment of osteoblasts with a Src family kinase inhibitor, PP2, or the expression of two independent kinase-dead Src mutant constructs caused significant inhibition of TGF-beta1 induced CTGF promoter activity and expression. Additionally, blocking Src activation prevented Erk activation by TGF-beta1 demonstrating a role for Src as an upstream mediator of Erk in regulating CTGF expression in osteoblasts. To investigate the involvement of the TGF-beta1 response element (TRE) and the SMAD binding element (SBE) in CTGF induction, we cloned the rat CTGF proximal promoter (-787 to +1) containing the TRE and SBE motifs into a pGL3-Luciferase reporter construct. Using a combination of CTGF promoter deletion constructs and site-directed mutants, we demonstrated the unique requirement of both the TRE and SBE for CTGF induction by TGF-beta1 in osteoblasts. Electro-mobility shift assays using specific probes containing the TRE, SBE or both showed TGF-beta1 inducible complexes that can be ablated by mutation of the respective motif, confirming their requirement for TGF-beta1 induced CTGF promoter activity. In conclusion, these studies demonstrate that CTGF induction by TGF-beta1 in osteoblasts involves Smads 3 and 4, the Erk and Src signaling pathways, and requires both the TRE and SBE motifs in the CTGF proximal promoter.

Connective tissue growth factor (CTGF/CCN2) is a downstream mediator for TGF-beta1-induced extracellular matrix production in osteoblasts.

Connective tissue growth factor (CTGF/CCN2) is a cysteine-rich, extracellular matrix (ECM) protein that acts as an anabolic growth factor to regulate osteoblast differentiation and function. Recent studies have identified CTGF as a downstream effector of transforming growth factor-beta1 (TGF-beta1) for certain functions in specific cell types. In this study, we examined the role of CTGF as a downstream mediator of TGF-beta1-induced ECM production and cell growth in osteoblasts. Using primary cultures, we demonstrated that TGF-beta1 is a potent inducer of CTGF expression in osteoblasts, and that this induction occurred at all stages of osteoblast differentiation from the proliferative through mineralization stages. TGF-beta1 treatment of osteoblasts increased the expression and synthesis of the ECM components, collagen and fibronectin. When CTGF-specific siRNA was used to prevent TGF-beta1 induction of CTGF expression, it also inhibited collagen and fibronectin production, thereby demonstrating the requirement of CTGF for their up-regulation. To examine the effects of TGF-beta1 on osteoblast cell growth, cultures were treated with TGF-beta1 during the proliferative stage. Cell number was significantly reduced and the cells exhibited a decrease in G1 cyclin expression, consistent with TGF-beta1-induced cell-cycle arrest. Cultures transfected with CTGF siRNA prior to TGF-beta1 treatment showed an even greater reduction in cell number, suggesting that TGF-beta1-induced growth arrest is independent of CTGF in osteoblasts. Collectively, these data demonstrate for the first time that CTGF is an essential downstream mediator for TGF-beta1-induced ECM production in osteoblasts, but these two growth factors function independently regarding their opposing effects on osteoblast proliferation.

Identification and characterization of the genes encoding human and mouse osteoactivin.

Osteoactivin (OA) is more highly expressed in the bones of osteopetrotic mutant rats (op/op) than in those of their normal littermates and is the homologue of human nmb, a cDNA more highly expressed in melanoma-derived cell lines of low metastatic potential, and of mouse DC-HIL, which has been implicated in endothelial cell adhesion. The human OA gene is found on chromosome 7p15.1 and consists of 11 exons spanning 28.3 kb. Murine OA is encoded by a highly similar gene of 11 exons spanning 20.2 kb on mouse chromosome 6. Human OA uses the same transcriptional initiation site in both bone and kidney as was reported for melanoma cells. OA is expressed in primary human and mouse osteoblast cultures at all stages of differentiation, with increased levels observed concurrently with the expression of osteoblast phenotype markers. OA is also expressed in a wide variety of human and mouse tissues as determined by RT-PCR analysis. Immunohistochemical investigation of OA expression in late mouse embryonic development showed very high, cell-specific expression in the nervous system, basal layer of the skin, germinal cells of hair follicles, and in the forming nephrons of the kidney. Continuing investigation of the cell-specific expression of OA in bone as well as in other tissues will lead to a better understanding of its function in the development of these cell types.

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.

Evidence that the rat osteopetrotic mutation toothless (tl) is not in the TNFSF11 (TRANCE, RANKL, ODF, OPGL) gene.

The toothless (tl) osteopetrotic mutation in the rat affects an osteoblast-derived factor that is required for normal osteoclast differentiation. Although the genetic locus remains unknown, the phenotypic impact of the tl mutation on multiple systems has been well characterized. Some of its actions are similar to tumornecrosis factor superfamily member 11(TNFSF11; also called TRANCE, RANKL, ODF and OPGL) null mice. TNFSF11 is a recently described member of the tumor necrosis factor superfamily which, when expressed by activated T cells, enhances the survival of antigen-presenting dendritic cells, and when expressed by osteoblasts, promotes the differentiation and activation of osteoclasts. The skeletal similarities between tl rats and TNFSF11(-/-) mice include 1) profound osteoclastopenia (TNFSF11-null mice, 0% and tl rats 0-1% of normal); 2) persistent, non-resolving osteopetrosis that results from 3) a defect not in the osteoclast lineage itself, but in an osteoblast-derived, osteoclastogenic signal; and 4) a severe chondrodysplasia of the growth plates of long bones not seen in other osteopetrotic mutations. The latter includes thickening of the growth plate with age, disorganization of chondrocyte columns, and disturbances of chondrocyte maturation. These striking similarities prompted us to undertake studies to rule in or out a TNFSF11 mutation in the tl rat. We looked for expression of TNFSF11 mRNA in tl long bones and found it to be over-expressed and of the correct size. We also tested TNFSF11 protein function in the tl rat. This was shown to be normal by flow cytometry experiments in which activated, spleen-derived T-cells from tl rats exhibited normal receptor binding competence, as measured by a recombinant receptor assay. We also found that tl rats develop histologically normal mesenteric and peripheral lymph nodes, which are absent from TNFSF11-null mice. Next, we found that injections of recombinant TNFSF11, which restores bone resorption in null mice, had no therapeutic effect in tl rats. Finally, gene mapping studies using co-segregation of polymorphic markers excluded the chromosomal region containing the TNFSF11 gene as harboring the mutation responsible for the tl phenotype. We conclude that, despite substantial phenotypic similarities to TNFSF11(-/-) mice, the tl rat mutation is not in the TNFSF11 locus, and that its identification must await the results of further studies.

A pollen-specific novel calmodulin-binding protein with tetratricopeptide repeats.

Calcium is essential for pollen germination and pollen tube growth. A large body of information has established a link between elevation of cytosolic Ca(2+) at the pollen tube tip and its growth. Since the action of Ca(2+) is primarily mediated by Ca(2+)-binding proteins such as calmodulin (CaM), identification of CaM-binding proteins in pollen should provide insights into the mechanisms by which Ca(2+) regulates pollen germination and tube growth. In this study, a CaM-binding protein from maize pollen (maize pollen calmodulin-binding protein, MPCBP) was isolated in a protein-protein interaction-based screening using (35)S-labeled CaM as a probe. MPCBP has a molecular mass of about 72 kDa and contains three tetratricopeptide repeats (TPR) suggesting that it is a member of the TPR family of proteins. MPCBP protein shares a high sequence identity with two hypothetical TPR-containing proteins from Arabidopsis. Using gel overlay assays and CaM-Sepharose binding, we show that the bacterially expressed MPCBP binds to bovine CaM and three CaM isoforms from Arabidopsis in a Ca(2+)-dependent manner. To map the CaM-binding domain several truncated versions of the MPCBP were expressed in bacteria and tested for their ability to bind CaM. Based on these studies, the CaM-binding domain was mapped to an 18-amino acid stretch between the first and second TPR regions. Gel and fluorescence shift assays performed with CaM and a CaM-binding synthetic peptide further confirmed MPCBP binding to CaM. Western, Northern, and reverse transcriptase-polymerase chain reaction analysis have shown that MPCBP expression is specific to pollen. MPCBP was detected in both soluble and microsomal proteins. Immunoblots showed the presence of MPCBP in mature and germinating pollen. Pollen-specific expression of MPCBP, its CaM-binding properties, and the presence of TPR motifs suggest a role for this protein in Ca(2+)-regulated events during pollen germination and growth.

The kinesin-like calmodulin binding protein is differentially involved in cell division.

The kinesin-like calmodulin (CaM) binding protein (KCBP), a minus end-directed microtubule motor protein unique to plants, has been implicated in cell division. KCBP is negatively regulated by Ca(2)+ and CaM, and antibodies raised against the CaM binding region inhibit CaM binding to KCBP in vitro; therefore, these antibodies can be used to activate KCBP constitutively. Injection of these antibodies into Tradescantia virginiana stamen hair cells during late prophase induces breakdown of the nuclear envelope within 2 to 10 min and leads the cell into prometaphase. However, mitosis is arrested, and the cell does not progress into anaphase. Injection of antibodies later during cell division has no effect on anaphase transition but causes aberrant phragmoplast formation and delays the completion of cytokinesis by approximately 15 min. These effects are achieved without any apparent degradation of the microtubule cytoskeleton. We propose that during nuclear envelope breakdown and anaphase, activated KCBP promotes the formation of a converging bipolar spindle by sliding and bundling microtubules. During metaphase and telophase, we suggest that its activity is downregulated.

Influence of estrogen deficiency and replacement on T-cell populations in rat lymphoid tissues and organs.

Estrogen deficiency following ovariectomy or menopause results in bone loss. Although evidence strongly suggests that the immune system is involved in the pathogenesis of estrogen-deficient osteoporosis, it is not clear what role, if any, the T-lymphocyte plays in this process. Therefore, we examined the distribution of T-cell subsets in lymphoid organs and tissues, under varying estrogenic states in the rat. Six-month-old female Sprague-Dawley rats, ovariectomized (Ovx) and sham-operated, were randomized 5 d post-surgery into six groups to receive the following treatments: (A) sham/placebo; (B) sham/low-dose E2; (C) sham/high-dose E2; (D) Ovx/placebo; (E) Ovx/low-dose E2; (F) Ovx/high-dose E2. Half of the treated rats (groups A-F) were sacrificed on d 14; the remainder on d 28. Following euthanasia, mononuclear cells were isolated from the thymus, peripheral blood, spleen, lymph node and bone marrow, and were labeled for flow cytometric analysis using mouse anti-rat monoclonal antibodies directed against CD5, CD4, and CD8 antigenic markers. In the thymus, ovariectomy caused a dramatic increase and E2 treatment caused a dose-dependent decrease in weight that was proportional to the number of thymocytes. In the bone marrow, ovariectomy caused a significant reduction in the percentage of all T-cell subsets examined and this effect persisted throughout the duration of the study. Estrogen replacement therapy at the low-dose reversed the effects of ovariectomy and high-dose E2 treatment caused an increase in T-cell subsets in both the sham and Ovx groups, an effect that was more pronounced at d 14 compared with d 28. Although the percentages of some T-cell subsets in the other lymphoid organs/tissues were altered by ovariectomy or E2 treatment at d 0 and 14, all these changes had normalized by d 28 except for CD5 and CD4 cells in peripheral blood. In summary, with the exception of T-lymphocytes in the bone marrow, the effects of varying estrogenic states on T-cells were variable and transient. The influence of estrogen status on bone marrow T-lymphocytes suggests that these cells may play a role in mediating the effects of estrogen on bone turnover and warrant additional studies focusing on the functional role of T-cells in the bone marrow compartment.

Cloning the full-length cDNA for rat connective tissue growth factor: implications for skeletal development.

The mammalian osteopetroses represent a pathogenetically diverse group of skeletal disorders characterized by excess bone mass resulting from reduced osteoclastic bone resorption. Abnormalities involving osteoblast function and skeletal development have also been reported in many forms of the disease. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between op mutants and their normal littermates. RNA isolated from calvaria and long bones was used as a template for mRNA-differential display. Sequence information for one of the many cDNA that were selectively expressed in either normal or mutant bone suggested that it is the rat homologue of connective tissue growth factor (CTGF) previously cloned in the human, mouse, and other species. A consensus sequence was assembled from overlapping 5'-RACE clones and used to confirm the rat CTGF cDNA protein coding region. Northern blot analysis confirmed that this message was highly (8- to 10-fold) over-expressed in op versus normal bone; it was also upregulated in op kidney but none of the other tissues (brain, liver, spleen, thymus) examined. In primary rat osteoblast cultures, the CTGF message exhibits a temporal pattern of expression dependent on their state of differentiation. Furthermore, CTGF expression is regulated by prostaglandin E(2), a factor known to modulate osteoblast differentiation. Since members of the CTGF family regulate the expression of specific genes, such as collagen and fibronectin, we propose that CTGF may play a previously unreported role in normal skeletal modeling/remodeling. Its dramatic over-expression in the op mutant skeleton may be secondary to the uncoupling of bone resorption and bone formation resulting in dysregulation of osteoblast gene expression and function.

Interaction of a kinesin-like protein with calmodulin isoforms from Arabidopsis.

In Arabidopsis and other plants there are multiple calmodulin isoforms. However, the role of these isoforms in regulating the activity of target proteins is obscure. Here, we analyzed the interaction between a kinesin-like calmodulin-binding motor protein (Reddy, A. S. N., Safadi, F., Narasimhulu, S. B., Golovkin, M., and Hu, X. (1996) J. Biol. Chem. 271, 7052-7060) and three calmodulin isoforms (calmodulin-2, -4, and -6) from Arabidopsis using different approaches. Gel mobility and fluorescence shift assays revealed that the motor binds to all calmodulin isoforms in a calcium-dependent manner. Furthermore, all calmodulin isoforms were able to activate bovine calcium/calmodulin-dependent phosphodiesterase. However, the concentration of calmodulin-2 required for half-maximal activation of phosphodiesterase is 2- and 6-fold lower compared with calmodulin-4 and -6, respectively. The dissociation constants of the motor to calmodulin-2, -4, and -6 are 12.8, 27.0, and 27.8 nM, respectively, indicating that calmodulin-2 has 2-fold higher affinity for the motor than calmodulin-4 and -6. Similar results were obtained using another assay that involves the binding of (35)S-labeled calmodulin isoforms to the motor. The binding saturation curves of the motor with calmodulin isoforms have confirmed that calmodulin-2 has 2-fold higher affinity to the motor. However, the affinity of calmodulin-4 and -6 isoforms for the motor was about the same. Based on these studies, we conclude that all calmodulin isoforms bind to the motor protein but with different affinities.

The toothless osteopetrotic rat has a normal vitamin D-binding protein-macrophage activating factor (DBP-MAF) cascade and chondrodysplasia resistant to treatments with colony stimulating factor-1 (CSF-1) and/or DBP-MAF.

The osteopetrotic rat mutation toothless (tl) is characterized by little or no bone resorption, few osteoclasts and macrophages, and chondrodysplasia at the growth plates. Short-term treatment of tl rats with colony-stimulating factor-1 (CSF-1) has been shown to increase the number of osteoclasts and macrophages, producing dramatic resolution of skeletal sclerosis at some, but not all, sites. Defects in production of vitamin D-binding protein-macrophage activating factor (DBP-MAF) have been identified in two other independent osteopetrotic mutations of the rat (op and ia), and two in the mouse (op and mi), in which macrophages and osteoclasts can be activated by the administration of exogenous DBP-MAF. The present studies were undertaken to examine the histology and residual growth defects in tl rats following longer CSF-1 treatments, to investigate the possibility that exogenous DBP-MAF might act synergistically with CSF-1 to improve the tl phenotype, and to assess the integrity of the endogenous DBP-MAF pathway in this mutation. CSF-1 treatment-with or without DBP-MAF-induced resorption of metaphyseal bone to the growth plate on the marrow side, improved slightly but did not normalize long bone growth, and caused no improvement in the abnormal histology of the growth plate. Injections of lysophosphatidylcholine (lyso-Pc) to prime macrophage activation via the DBP-MAF pathway raised superoxide production to similar levels in peritoneal macrophages from both normal and mutant animals, indicating no defect in the DBP-MAF pathway in tl rats. Interestingly, pretreatments with CSF-1 alone also increased superoxide production, although the mechanism for this remains unknown. In summary, we find that, unlike other osteopetrotic mutations investigated to date, the DBP-MAF pathway does not appear to be defective in the tl rat; that additional DBP-MAF does not augment the beneficial skeletal effects seen with CSF-1 alone; and that the growth plate chondrodystrophy seen in this mutation is unaffected by either molecule. Thus, the tl mutation intercepts the function of a gene required for both normal endochondral ossification and bone resorption, thereby uncoupling the coordination of skeletal metabolism required for normal long bone growth.

Osteopathy and resistance to vitamin D toxicity in mice null for vitamin D binding protein.

A line of mice deficient in vitamin D binding protein (DBP) was generated by targeted mutagenesis to establish a model for analysis of DBP's biological functions in vitamin D metabolism and action. On vitamin D-replete diets, DBP-/- mice had low levels of total serum vitamin D metabolites but were otherwise normal. When maintained on vitamin D-deficient diets for a brief period, the DBP-/-, but not DBP+/+, mice developed secondary hyperparathyroidism and the accompanying bone changes associated with vitamin D deficiency. DBP markedly prolonged the serum half-life of 25(OH)D and less dramatically prolonged the half-life of vitamin D by slowing its hepatic uptake and increasing the efficiency of its conversion to 25(OH)D in the liver. After an overload of vitamin D, DBP-/- mice were unexpectedly less susceptible to hypercalcemia and its toxic effects. Peak steady-state mRNA levels of the vitamin D-dependent calbindin-D9K gene were induced by 1,25(OH)2D more rapidly in the DBP-/- mice. Thus, the role of DBP is to maintain stable serum stores of vitamin D metabolites and modulate the rates of its bioavailability, activation, and end-organ responsiveness. These properties may have evolved to stabilize and maintain serum levels of vitamin D in environments with variable vitamin D availability.

Skeletal resistance to 1,25-dihydroxyvitamin D3 in osteopetrotic rats.

The osteopetrotic (op/op) rat mutation is a lethal mutation in which decreased osteoclast function (bone resorption) coexists with markedly elevated serum levels of 1 ,25-dihydroxyvitamin D3[1,25(OH)2D3]. Increased circulating levels of 1,25(OH)2D3 have been reported in other osteopetrotic animal mutations and in some osteopetrotic children. This study examined the effects of 1,25(OH)2D3 infusions on serum and skeletal parameters in normal and mutant rats of op stock. We also examined vitamin D receptor expression and binding in bone cells from op normal and mutant animals. Four-week-old normal and mutant rats were infused either with propylene glycol (used as controls) or with 12.5-125 ng of 1,25(OH)2D3/d using osmotic minipumps implanted subcutaneously for 1 wk. Sera were analyzed for calcium, phosphorus, and 1,25(OH)2D3 levels. Histomorphometric analyses of proximal tibiae from treated normal (50 ng/d) and op mutant (125 ng/d) rats and their vehicle-infused controls were performed. Normal animals infused with 1,25(OH)2D3 exhibited a dose-dependent increase in serum calcium levels. Histomorphometric analyses of metaphyseal bone within the primary spongiosae region showed that 1,25(OH)2D3 increased osteoclast number with a reduction in osteoblast surface associated with a decrease in growth plate cartilage thickness. However, similar analyses on secondary spongiosae showed a decrease in osteoclast number and surface associated with an anabolic response. Op mutants infused with 1,25(OH)2D3 did not exhibit any change in serum calcium levels or histomorphometric parameters related to growth plate cartilage and metaphyseal bone compared with mutant controls. Vitamin D mRNA and protein levels were increased twoto threefold in op mutants compared to age-matched normal rats. However, binding affinity of 1,25(OH)2D3 to its receptor was similar between op mutant and normal animals. High dose calcitriol therapy, under the conditions and period of treatment used in this study, failed to stimulate bone turnover in op rats, suggesting that they are resistant to the skeletal effects of 1,25(OH)2D3. The failure of osteoclast activation in response to 1,25(OH)2D3 treatment may be associated with osteoblast incompetence in this mutation.

Partial purification and characterization of a Ca(2+)-dependent proteinase from Arabidopsis roots.

Ca2+, an important intracellular messenger in plants, is implicated in controlling diverse cellular functions by regulating the activity of several enzymes. Here we report the presence of a Ca(2+)-dependent proteinase (CDP) activity in roots of Arabidopsis using in-gel assays (zymograms). The CDP activity showed absolute Ca2+ requirement for its activation; other divalent ions such as Mg2+, Sr2+, and Zn2+ did not substitute for Ca2+ in stimulating protease activity. The CDP activity was inhibited by the proteinase inhibitors leupeptin, E-64, and N-ethylmaleimide, whereas pepstatin A and phenylmethylsulfonyl fluoride were without effect. These data indicate that the enzyme is likely to be a cysteine proteinase. The CDP activity was partially purified from root cultures using ammonium sulfate precipitation, DE-52, Mono-Q, and Superdex 200 column chromatography. This purification scheme resulted in about 40-fold purification of the CDP activity. Based on the elution of Arabidopsis CDP (ACDP) activity on gel filtration column the molecular mass of CDP was estimated to be about 75 kDa. Isoelectric focusing showed that the enzyme had a pI between 5.2 and 5.4. SDS-polyacrylamide gel analysis showed that activity was associated with a 45-kDa polypeptide, suggesting that the native ACDP is a homodimer. Five different antibodies raised to animal CDPs did not cross-react with the partially purified protein. These data suggest that the plant CDP differs from the known CDPs characterized from animals and is likely to be a new CDP that is unique to plants.

A plant kinesin heavy chain-like protein is a calmodulin-binding protein.

Calmodulin, a calcium modulated protein, regulates the activity of several proteins that control cellular functions. A cDNA encoding a unique calmodulin-binding protein, PKCBP, was isolated from a potato expression library using protein-protein interaction based screening. The cDNA encoded protein bound to biotinylated calmodulin and 35S-labeled calmodulin in the presence of calcium and failed to bind in the presence of EGTA, a calcium chelator. The deduced amino acid sequence of the PKCBP has a domain of about 340 amino acids in the C-terminus that showed significant sequence similarity with the kinesin heavy chain motor domain and contained conserved ATP- and microtubule-binding sites present in the motor domain of all known kinesin heavy chains. Outside the motor domain, the PKCBP showed no sequence similarity with any of the known kinesins, but contained a globular domain in the N-terminus and a putative coiled-coil region in the middle. The calmodulin-binding region was mapped to a stretch of 64 amino acid residues in the C-terminus region of the protein. The gene is differentially expressed with the highest expression in apical buds. A homolog of PKCBP from Arabidopsis (AKCBP) showed identical structural organization indicating that kinesin heavy chains that bind to calmodulin are likely to exist in other plants. This paper presents evidence that the motor domain has microtubule stimulated ATPase activity and binds to microtubules in a nucleotide-dependent manner. The kinesin heavy chain-like calmodulin-binding protein is a new member of the kinesin superfamily as none of the known kinesin heavy chains contain a calmodulin-binding domain. The presence of a calmodulin-binding motif and a motor domain in a single polypeptide suggests regulation of kinesin heavy chain driven motor function(s) by calcium and calmodulin.