John R. Harrison

A New Kv1.2 Channelopathy Underlying Cerebellar Ataxia

A forward genetic screen of mice treated with the mutagen ENU identified a mutant mouse with chronic motor incoordination. This mutant, named Pingu (Pgu), carries a missense mutation, an I402T substitution in the S6 segment of the voltage-gated potassium channel Kcna2. The gene Kcna2 encodes the voltage-gated potassium channel α-subunit Kv1.2, which is abundantly expressed in the large axon terminals of basket cells that make powerful axo-somatic synapses onto Purkinje cells. Patch clamp recordings from cerebellar slices revealed an increased frequency and amplitude of spontaneous GABAergic inhibitory postsynaptic currents and reduced action potential firing frequency in Purkinje cells, suggesting that an increase in GABA release from basket cells is involved in the motor incoordination in Pgu mice. In line with immunochemical analyses showing a significant reduction in the expression of Kv1 channels in the basket cell terminals of Pgu mice, expression of homomeric and heteromeric channels containing the Kv1.2(I402T) α-subunit in cultured CHO cells revealed subtle changes in biophysical properties but a dramatic decrease in the amount of functional Kv1 channels. Pharmacological treatment with acetazolamide or transgenic complementation with wild-type Kcna2 cDNA partially rescued the motor incoordination in Pgu mice. These results suggest that independent of known mutations in Kcna1 encoding Kv1.1, Kcna2 mutations may be important molecular correlates underlying human cerebellar ataxic disease.

Impaired Cortical Bone Acquisition and Osteoblast Differentiation in Mice with Osteoblast-Targeted Disruption of Glucocorticoid Signaling

To determine the role of endogenous glucocorticoids in bone, we previously developed transgenic mice in which a 2.3 kb fragment of the Col1a1 promoter drives 11ß-hydroxysteroid dehydrogenase 2 expression in mature osteoblasts. This transgene should inactivate glucocorticoids upstream of all receptor signaling pathways. In the present study, we show that femoral cortical bone area and thickness were approximately 10–15% lower in transgenic mice than in wild-type littermates. Femur length was unchanged, indicating that bone elongation was not affected in this model. Expression of osteocalcin mRNA, pOBCol2.3-GFP (a green fluorescent protein marker of mature osteoblasts), and the formation of mineralized nodules were impaired in ex vivo transgenic primary calvarial cultures. The extent of crystal violet staining in bone marrow cultures, indicative of the number of adherent stromal cells, was also decreased. These data suggest that endogenous glucocorticoids are required for cortical bone acquisition and full osteoblast differentiation. It appears that blocking glucocorticoid signaling in vivo leads to a decrease in the commitment and/or expansion of progenitors entering the osteoblast lineage.

Superfluous Role of Mammalian Septins 3 and 5 in Neuronal Development and Synaptic Transmission

ABSTRACT The septin family of GTPases, first identified for their roles in cell division, are also expressed in postmitotic tissues. SEPT3 (G-septin) and SEPT5 (CDCrel-1) are highly expressed in neurons, enriched in presynaptic terminals, and associated with synaptic vesicles. These characteristics suggest that SEPT3 or SEPT5 might be important for synapse formation, maturation, or synaptic vesicle traffic. Since Sept5−/− mice do not show any overt neurological phenotypes, we generated Sept3−/− and Sept3−/−Sept5−/− mice and found that SEPT3 and SEPT5 are not essential for development, fertility, or viability. Changes in the expression of septins were noted in the absence of SEPT3, SEPT5, and both septins. SEPT5 association with other septins in brain tissue was unaffected by the removal of SEPT3. No abnormalities were observed in the gross morphology and synapses of the hippocampus. Similarly, axon development and synapse formation were unaffected in vitro. In cultured hippocampal neurons, the size of the recycling synaptic vesicle pool was unaltered in the absence of SEPT3. Furthermore, synaptic transmission at two different central synapses was not significantly affected in Sept3−/−Sept5−/− mice. These results indicate that SEPT3 and SEPT5 are dispensable for neuronal development as well as for synaptic vesicle fusion and recycling.

Identification of multiple cis-acting elements mediating the induction of prostaglandin G/H synthase-2 by phorbol ester in murine osteoblastic cells.

The tumor promoter phorbol 13‐myristate 12‐acetate (PMA), the best characterized protein kinase C agonist, frequently regulates gene expression via activation of Fos/Jun (AP‐1) complexes. PMA rapidly and transiently induces prostaglandin G/H synthase‐2 (PGHS‐2) expression in murine osteoblastic MC3T3‐E1 cells, but no functional AP‐1 binding motifs in the 5′‐flanking region have been identified. In MC3T3‐E1 cells transfected with −371/+70 bp of the PGHS‐2 gene fused to a luciferase reporter gene (Pluc), PMA stimulates luciferase activity up to eightfold. Computer analysis of the sequence of the PGHS‐2 promoter region identified three potential AP‐1 elements in the −371/+70 bp region, and deletion analysis suggested that the sequence 5′‐aGAGTCA‐3′ at −69/−63 bp was most likely to mediate stimulation by PMA. Mutation of the putative AP‐1 sequence reduces the ability of PMA to stimulate Pluc activity by 65%. On electrophoretic mobility shift analysis (EMSA), PMA induces binding to a PGHS‐2 probe spanning this sequence, binding is blocked by an unlabeled AP‐1 canonical sequence, and antibodies specific for c‐Jun and c‐Fos inhibit binding. Mutation of this AP‐1 site also causes a small (22%) but significant reduction in the serum stimulation of Pluc activity in transiently transfected MC3T3‐E1 cells. On EMSA, serum induces binding to a PGHS‐2 probe spanning the AP‐1 site, binding is blocked by an unlabeled AP‐1 canonical sequence, and antibodies specific for c‐Jun and c‐Fos inhibit binding. Joint mutation of this AP‐1 site and the nearby CRE site at −56/−52 bp, previously shown to mediate serum, v‐src and PDGF induction of PGHS‐2 in NIH‐3T3 cells, blocks both PMA and serum induction of Pluc activity in MC3T3‐E1 cells. Hence, the AP‐1 and CRE binding sites are jointly but differentially involved in both the PMA and serum stimulation of PGHS‐2 promoter activity. J. Cell. Biochem. 78:197–209, 2000.

Cloning and in Vitro Characterization ofα 1(I)-Collagen 11β-Hydroxysteroid Dehydrogenase Type 2 Transgenes as Models for Osteoblast-Selective Inactivation of Natural Glucocorticoids1

The NAD-dependent enzyme, 11β-hydroxysteroid dehydrogenase type II (11βHSD2), catalyzes the unidirectional conversion of biologically active glucocorticoids to inactive metabolites. In vivo, 11βHSD2 protects the mineralocorticoid receptor from activation by glucocorticoids in mineralocorticoid target tissues such as kidney. The goal of the present study was to use targeted overexpression of 11βHSD2 as a novel means of disrupting glucocorticoid signaling in osteoblastic cells. Rat 11βHSD2 complementary DNA was cloned downstream of a 2.3- and 3.6-kb α1(I)-collagen (Col1a1) promoter fragment to produce the expression plasmids Col2.3-HSD2 and Col3.6-HSD2, respectively, which were transiently and/or stably transfected in osteoblastic ROS 17/2.8 and MC3T3-E1 cells. Transgene messenger RNA and protein were detected in transfected cells by Northern blot analysis and immunostaining, respectively. Transfection of 11βHSD2 led to higher rates of conversion of[ 3H]corticosterone to[ 3H]dehydrocorticosterone and reduce...

Involvement of CCAAT Enhancer Binding Protein Transcription Factors in the Regulation of Prostaglandin G/H Synthase 2 Expression by Interleukin-1 in Osteoblastic MC3T3-E1 Cells

Interleukin‐1 (IL‐1) stimulates prostaglandin production in bone by a rapid and transient activation of prostaglandin G/H synthase 2 (PGHS‐2) gene expression. In osteoblastic MC3T3‐E1 cells, IL‐1 caused a transient increase in PGHS‐2 messenger RNA (mRNA), which peaked at 2 h. IL‐1 caused a 2‐ to 4‐fold activation of a 371‐base pair (bp) murine PGHS‐2 promoter/luciferase construct in stable transfectants. This response mapped to a proximal promoter element(s) located between −150 and −40 bp. This region contains a putative CCAAT enhancer binding protein (C/EBP) site (centered at −135 bp), which shows enhanced binding of C/EBPβ and C/EBPδ by mobility shift analysis after IL‐1 treatment. A transient cotransfection approach was used to examine the effects of C/EBPβ and C/EBPδ overexpression. IL‐1 caused a maximal 3‐ to 7‐fold stimulation of PGHS‐2 promoter activity after 2.5 h. Overexpression of murine C/EBPβ and C/EBPδ caused a dose‐dependent increase in basal and IL‐1‐stimulated luciferase activity. C/EBPδ caused a greater enhancement of basal and IL‐1‐stimulated promoter activity than C/EBPβ, suggesting that C/EBPδ is a stronger transactivator. Overexpression of p20C/EBPβ, a dominant negative inhibitor of C/EBP function, blocked the stimulation of PGHS‐2 promoter activity by IL‐1 and blocked the ability of overexpressed C/EBPβ and C/EBPδ to increase basal and IL‐1‐stimulated promoter activity. Mutagenesis of the C/EBP site reduced, but did not abolish, the stimulation of PGHS‐2 promoter activity by IL‐1 and blunted the effect of overexpressed C/EBPδ on basal and IL‐1‐stimulated promoter activity. These results suggest an essential role for C/EBPβ and C/EBPδ in the induction of PGHS‐2 gene expression by IL‐1 in osteoblastic cells.

Avian medullary bone in organ culture: effects of vitamin D metabolites on collagen synthesis.

SummaryA new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of3H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10–15% of the total protein labeled. The addition of 1,25-dihydroxycholecalciferol (1,25 (OH)2D3) resulted in a dose-dependent inhibition of3H-proline incorporation into CDP at doses from 10−10M to 10−7M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since3H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)2D3-induced decrease in the3H-hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)2D3 is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaiored incorporation of collagen into bone matrix, or bone resorption Medullary bone collagen synthesis was not affected by 24,25(OH)2D3, either alone or in combination with 1,25(OH)2D3. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)2D3 and 1,24,25(OH)3D3 were the most potent metabolites tested, followed by 1-alpha(OH)D3. 25(OH)D3 and 24,25(OH)2D3 had no effect at concentrations as high as 10−7M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as anin vitro model for the study of bone formation

Osteopenia in transgenic mice with osteoblast-targeted expression of the inducible cAMP early repressor

ICER is a member of the CREM family of basic leucine zipper transcription factors that acts as a dominant negative regulator of gene transcription. Four different isoforms of ICER (I, Igamma, II and IIgamma) are transcribed from the P2 promoter of the Crem gene. We previously found that each of the ICER isoforms is induced by parathyroid hormone in osteoblasts. The goal of the present study was to assess the function of ICER in bone by overexpressing ICER in osteoblasts of transgenic mice. ICER I and ICER II cDNAs, each containing an N-terminal FLAG epitope tag, were cloned downstream of a fragment containing 3.6 kb of the rat Col1a1 promoter and most of the rat Col1a1 first intron to produce pOBCol3.6-ICER I and pOBCol3.6-ICER II transgenes, respectively. Multiple lines of mice were generated bearing the ICER I and ICER II transgenes. At 8 weeks of age, ICER I and ICER II transgenic mice had lower body weights and decreased bone mineral density of femurs and vertebrae. Further studies were done with ICER I transgenic mice, which had greatly reduced trabecular bone volume and a markedly decreased bone formation rate in femurs. Osteoblast differentiation and osteocalcin expression were reduced in ex vivo bone marrow cultures from ICER I transgenic mice. ICER I antagonized the activity of ATF4 at its consensus DNA binding site in the osteocalcin promoter in vitro. Thus, transgenic mice with osteoblast-targeted overexpression of ICER exhibited osteopenia caused primarily by reduced bone formation. We speculate that ICER regulates the activity and/or expression of ATF/CREB factors required for normal bone formation.

Interleukin‐1 Represses COLIA1 Promoter Activity in Calvarial Bones of Transgenic ColCAT Mice In Vitro and In Vivo

Interleukin‐1 (IL‐1) inhibits collagen synthesis in osteoblastic cell lines and primary osteoblast‐like cells. However, promoter elements regulating type I collagen A1 (COLIA1) expression in vivo and in organ culture may differ from those regulating expression in cell culture. We have examined the effects of IL‐1 on reporter gene activity in neonatal transgenic mouse calvariae bearing COLIA1 promoter‐chloramphenicol acetyltransferase (ColCAT) fusion genes. The parent construct, ColCAT 3.6, contains 3.5 kb of 5′ flanking sequence and 115 bp of 5′ untranslated region fused to the CAT reporter. In 48‐h calvarial organ cultures, IL‐1 repressed ColCAT 3.6 promoter activity and collagen synthesis in a dose‐related manner, with a maximal inhibition of 40–65%. This repression was retained in 5′ deletion constructs truncated to −1719 bp. The inhibition of transgene mRNA was blocked by cycloheximide, indicating a requirement for new protein synthesis. Pretreatment with indomethacin diminished the inhibitory effect of IL‐1 on CAT activity and collagen synthesis, suggesting partial mediation by prostaglandins. Local in vivo injection of IL‐1 (500 ng) decreased calvarial transgene mRNA after 8 h, an effect that was partially blocked by indomethacin. ColCAT transgenic mice represent a useful model for in vitro and in vivo assessment of COLIA1 promoter regulation by cytokines and other factors.

Col3.6-HSD2 transgenic mice: A glucocorticoid loss-of-function model spanning early and late osteoblast differentiation

The goal of this study was to characterize the bone phenotype and molecular alterations in Col3.6-HSD2 mice in which a 3.6-kb Col1a1 promoter fragment drives 11beta-HSD2 expression broadly in the osteoblast lineage to reduce glucocorticoid signaling. Serum corticosterone was unchanged in transgenic females excluding a systemic effect of the transgene. Adult transgenic mice showed reduced vertebral trabecular bone volume and reduced femoral and tibial sub-periosteal and sub-endosteal areas as assessed by microCT. In adult female transgenic mice, histomorphometry showed that vertebral bone mass and trabecular number were reduced but that osteoblast and osteoclast numbers and the mineral apposition and bone formation rates were not changed, suggesting a possible developmental defect in the formation of trabeculae. In a small sample of male mice, osteoblast number and percent osteoid surface were increased but the mineral apposition bone formation rates were not changed, indicating subtle sex-specific phenotypic differences in Col3.6-HSD2 bone. Serum from transgenic mice had decreased levels of the C-terminal telopeptide of alpha1(I) collagen but increased levels of osteocalcin. Transgenic calvarial osteoblast and bone marrow stromal cultures showed decreased alkaline phosphatase and mineral staining, reduced levels of Col1a1, bone sialoprotein and osteocalcin mRNA expression, and decreased cell growth and proliferation. Transgenic bone marrow cultures treated with RANKL and M-CSF showed greater osteoclast formation; however, osteoclast activity as assessed by resorption of a calcium phosphate substrate was decreased in transgenic cultures. Gene profiling of cultured calvarial osteoblasts enriched in the Col3.6-HSD2 transgene showed modest but significant changes in gene expression, particularly in cell cycle and integrin genes. In summary, Col3.6-HSD2 mice showed a low bone mass phenotype, with decreased ex vivo osteogenesis. These data further strengthen the concept that endogenous glucocorticoid signaling is required for optimal bone mass acquisition and highlight the complexities of glucocorticoid signaling in bone cell lineages.