Genetic and pharmacologic modulation of cementogenesis via pyrophosphate regulators.

Pyrophosphate (PPi) serves as a potent and physiologically important regulator of mineralization, with systemic and local concentrations determined by several key regulators, including: tissue-nonspecific alkaline phosphatase (ALPL gene; TNAP protein), the progressive ankylosis protein (ANKH; ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1; ENPP1). Results to date have indicated important roles for PPi in cementum formation, and we addressed several gaps in knowledge by employing genetically edited mouse models where PPi metabolism was disrupted and pharmacologically modulating PPi in a PPi-deficient mouse model. We demonstrate that acellular cementum growth is inversely proportional to PPi levels, with reduced cementum in Alpl KO (increased PPi levels) mice and excess cementum in Ank KO mice (decreased PPi levels). Moreover, simultaneous ablation of Alpl and Ank results in reestablishment of functional cementum in dKO mice. Additional reduction of PPi by dual deletion of Ank and Enpp1 does not further increase cementogenesis, and PDL space is maintained in part through bone modeling/remodeling by osteoclasts. Our results provide insights into cementum formation and expand our knowledge of how PPi regulates cementum. We also demonstrate for the first time that pharmacologic manipulation of PPi through an ENPP1-Fc fusion protein can regulate cementum growth, supporting therapeutic interventions targeting PPi metabolism.

AP2alpha alters the transcriptional activity and stability of p53.

AP2alpha and p53 form nuclear complexes that establish a functional partnership, which regulates the expression of certain genes involved in cell growth and metastasis. The growth effects of AP2alpha are mediated through p21WAF1/CIP1 and the ability for AP2alpha to coactivate p21 requires p53. Herein, we have localized the AP2-binding region of p53 to amino acids 305-375. Analysis of 26 distinct p53 alleles established a correlation between AP2alpha binding and transcriptional coactivation. The L350P point mutation was the only nonbinding allele that retained normal transcriptional activity by reporter assay. Although both wild-type and L350P alleles facilitated binding of AP2alpha to the p21 promoter, the L350P allele was significantly reduced in its ability to induce the endogenous p21 gene, demonstrating a striking difference in activity comparing reporter assays with activation of endogenous p53 target genes. Interestingly, expression of AP2 in the absence of radiation repressed p53-mediated induction of p21 and this effect was explained by a reduction in p53 stability induced by AP2alpha overexpression. We conclude that AP2alpha has competing effects on p53 activity through coactivation and decreased stability. These findings may provide a mechanism to account for the discrepancies reported for the association between AP2 and p21 expression in tumor tissue.

Identification and characterization of beta V spectrin, a mammalian ortholog of Drosophila beta H spectrin.

Four mammalian beta-spectrin genes are currently recognized, all encode proteins of approximately 240-280,000 M(r) and display 17 triple helical homologous approximately 106-residue repeat units. In Drosophila and Caenorhabditis elegans, a variant beta spectrin with unusual properties has been recognized. Termed beta heavy (beta(H)), this spectrin contains 30 spectrin repeats, has a molecular weight in excess of 400,000, and associates with the apical domain of polarized epithelia. We have cloned and characterized from a human retina cDNA library a mammalian ortholog of Drosophila beta(H) spectrin, and in accord with standard spectrin naming conventions we term this new mammalian spectrin beta 5 (betaV). The gene for human betaV spectrin (HUBSPECV) is on chromosome 15q21. The 11, 722-nucleotide cDNA of betaV spectrin is generated from 68 exons and is predicted to encode a protein with a molecular weight of 416,960. Like its fly counterpart, the derived amino acid sequence of this unusual mammalian spectrin displays 30 spectrin repeats, a modestly conserved actin-binding domain, a conserved membrane association domain 1, a conserved self-association domain, and a pleckstrin homology domain near its COOH terminus. Its putative ankyrin-binding domain is poorly conserved and may be inactive. These structural features suggest that betaV spectrin is likely to form heterodimers and oligomers with alpha spectrin and to interact directly with cellular membranes. Unlike its Drosophila ortholog, betaV spectrin does not contain an SH3 domain but displays in repeat 5 a 45-residue insertion that displays 42% identity to amino acids 85-115 of the E4 protein of type 75 human papilloma virus. Human betaV spectrin is expressed at low levels in many tissues. By indirect immunofluorescence, it is detected prominently in the outer segments of photoreceptor rods and cones and in the basolateral membrane and cytosol of gastric epithelial cells. Unlike its Drosophila ortholog, a distinct apical distribution of betaV spectrin is inapparent in the epithelial cell populations examined, although it is confined to the outer segments of photoreceptor cells. The complete cDNA sequence of human betaV spectrin is available from GenBank(TM) as accession number.

A widely expressed betaIII spectrin associated with Golgi and cytoplasmic vesicles.

Spectrin is an important structural component of the plasma membrane skeleton. Heretofore-unidentified isoforms of spectrin also associate with Golgi and other organelles. We have discovered another member of the beta-spectrin gene family by homology searches of the GenBank databases and by 5' rapid amplification of cDNA ends of human brain cDNAs. Collectively, 7,938 nucleotides of contiguous clones are predicted to encode a 271,294-Da protein, called betaIII spectrin, with conserved actin-, protein 4.1-, and ankyrin-binding domains, membrane association domains 1 and 2, a spectrin dimer self-association site, and a pleckstrin-homology domain. betaIII spectrin transcripts are concentrated in the brain and present in the kidneys, liver, and testes and the prostate, pituitary, adrenal, and salivary glands. All of the tested tissues contain major 9.0-kb and minor 11.3-kb transcripts. The human betaIII spectrin gene (SPTBN2) maps to chromosome 11q13 and the mouse gene (Spnb3) maps to a syntenic region close to the centromere on chromosome 19. Indirect immunofluorescence studies of cultured cells using antisera specific to human betaIII spectrin reveal a Golgi-associated and punctate cytoplasmic vesicle-like distribution, suggesting that betaIII spectrin associates with intracellular organelles. This distribution overlaps that of several Golgi and vesicle markers, including mannosidase II, p58, trans-Golgi network (TGN)38, and beta-COP and is distinct from the endoplasmic reticulum markers calnexin and Bip. Liver Golgi membranes and other vesicular compartment markers cosediment in vitro with betaIII spectrin. betaIII spectrin thus constitutes a major component of the Golgi and vesicular membrane skeletons.

ADP ribosylation factor regulates spectrin binding to the Golgi complex.

Homologues of two major components of the well-characterized erythrocyte plasma-membrane-skeleton, spectrin (a not-yet-cloned isoform, betaI Sigma* spectrin) and ankyrin (AnkG119 and an approximately 195-kDa ankyrin), associate with the Golgi complex. ADP ribosylation factor (ARF) is a small G protein that controls the architecture and dynamics of the Golgi by mechanisms that remain incompletely understood. We find that activated ARF stimulates the in vitro association of betaI Sigma* spectrin with a Golgi fraction, that the Golgi-associated betaI Sigma* spectrin contains epitopes characteristic of the betaI Sigma2 spectrin pleckstrin homology (PH) domain known to bind phosphatidylinositol 4,5-bisphosphate (PtdInsP2), and that ARF recruits betaI Sigma* spectrin by inducing increased PtdInsP2 levels in the Golgi. The stimulation of spectrin binding by ARF is independent of its ability to stimulate phospholipase D or to recruit coat proteins (COP)-I and can be blocked by agents that sequester PtdInsP2. We postulate that a PH domain within betaI Sigma* Golgi spectrin binds PtdInsP2 and acts as a regulated docking site for spectrin on the Golgi. Agents that block the binding of spectrin to the Golgi, either by blocking the PH domain interaction or a constitutive Golgi binding site within spectrin's membrane association domain I, inhibit the transport of vesicular stomatitis virus G protein from endoplasmic reticulum to the medial compartment of the Golgi complex. Collectively, these results suggest that the Golgi-spectrin skeleton plays a central role in regulating the structure and function of this organelle.

Utilization of an 86 bp exon generates a novel adducin isoform (beta 4) lacking the MARCKS homology domain.

A novel isoform of beta-adducin has been amplified and characterized from a human bone marrow cDNA library (GenBank #U43959). This isoform arises from the insertion of an 86 bp alternatively spliced and previously unrecognized exon (now termed exon 15) within codon 581 of the human red blood cell beta-adducin sequence. This results in an insertion of 28 novel amino acids. The remainder of the red cell beta-adducin mRNA is then translated in a different reading frame, adding an additional 35 novel amino acids prior to the stop codon. This new isoform, thus, replaces beta 1-adducin sequence after residue 580 with a total of 63 new amino acids. Sequences from genomic clones of the human beta-adducin gene show that this alternate exon is flanked by splice consensus sequences and is appropriately located in the genomic map between exons encoding up-stream and down-stream sequences, thus defining a new exon. The COOH-terminus of this new isoform, which we designate beta 4, lacks a 22 amino acid lysine-rich sequence common to both the human red cell alpha- and beta-adducin subunits and homologous to a highly conserved region in MARCKS, a filamentous actin-cross linking protein regulated by protein kinase C and calcium/calmodulin. beta 4-adducin preserves a previously identified calmodulin binding domain. PCR analysis indicates that this new beta-adducin isoform is expressed in fetal brain and liver, bone marrow, and NT-2 (neuroepithelial) cells, but is not detected in several other tissues. We anticipate that this new beta 4 isoform of beta-adducin will display unique and tissue-specific functional properties.

Na,K-ATPase transport from endoplasmic reticulum to Golgi requires the Golgi spectrin-ankyrin G119 skeleton in Madin Darby canine kidney cells.

Spectrin (betaISigma*) and ankyrin (AnkG119) associate with Golgi membranes and the dynactin complex, but their role in vesicle trafficking remains uncertain. We find that the actin-binding domain and membrane-association domain 1 (MAD1) of betaI spectrin together form a constitutive Golgi targeting signal in transfected MDCK cells. Expression of this signal in transfected cells disrupts the endogenous Golgi spectrin skeleton and blocks transport of alpha- and beta-Na,K-ATPase and vesicular stomatitis virus-G protein from the endoplasmic reticulum (ER) but does not disrupt the formation of Golgi stacks, the distribution of beta-COP, or the transport and surface display of E-cadherin. The Golgi spectrin skeleton is thus required for the transport of a subset of membrane proteins from the ER to the Golgi. We postulate that together with polyfunctional adapter proteins such as AnkG119, Golgi spectrin forms a docking complex that acts prior to the cis-Golgi, presumably with vesicular-tubular clusters (VTCs or ERGIC), to sequester specific membrane proteins into vesicles transiting between the ER and Golgi, and subsequently (probably involving other isoforms of spectrin and ankyrin) to mediate cargo transport within the Golgi and to other membrane compartments. We hypothesize that this vesicular spectrin-ankyrin adapter-protein trafficking (or tethering) system (SAATS) mediates the capture and transport of many membrane proteins and acts in conjunction with vesicle-targeting molecules to effect the efficient transport of cargo proteins.

Site-directed mutagenesis of alpha II spectrin at codon 1175 modulates its mu-calpain susceptibility.

Intracellular proteolysis by the calpains, a family of Ca2+ activated cysteine proteases, is a ubiquitous yet poorly understood process. Their action is implicated in an array of cellular and pathologic processes, including long-term potentiation, synaptic remodeling, protein kinase C and steroid receptor activation, ischemic cellular injury, and apoptosis. Unlike most proteases, the calpains display unusually strict substrate specificity, often cleaving only one or two bonds in proteins with hundreds of potential sites. Studies of synthetic peptides have defined sequences that modulate their specificity, but little data exist in the context of a bona fide protein. A prominent substrate for mu-calpain is alpha II spectrin (fodrin, brain spectrin), which is cleaved between Tyr1176 and Gly1177 within spectrin's 11th structural repeat unit. We have cloned and characterized human fetal brain alpha II spectrin (GenBank no. U26396) and identified a new Thr1300 to Ile polymorphism. From this clone, recombinant GST-fusion proteins representing repeat units 8-14 have been prepared and used to systematically explore the in vitro determinants of mu-calpain sensitivity. Twenty different amino acids were substituted by site-directed mutagenesis for wild-type Val1175, the penultimate (P2) residue flanking the major calpain cleavage site in alpha II spectrin. Gly, Pro, and Asp, and to a lesser extent Phe and Glu, substantively inhibited the susceptibility of this site to mu-calpain; other substitutions yielded lesser effects. Dynamic molecular modeling of the 11th structural repeat of human alpha II spectrin incorporating the various mutations suggests that the calpain cleavage site with its flanking calmodulin binding domain interrupts helix C of alpha II spectrin's 11th repetitive unit without significantly disrupting the repeat's triple-helical motif. This model predicts that the critical Tyr1176-Gly1177 bond occurs in a highly exposed loop juxtaposed between helix C and the calmodulin binding domain and that mutations at the P2 position subtly alter the conformation about this site. We conclude that secondary and tertiary conformational features surrounding the cleavage site, rather than the linear sequence itself, dominate the determinants that define alpha II spectrin's mu-calpain susceptibility.

The lethal hemolytic mutation in beta I sigma 2 spectrin Providence yields a null phenotype in neonatal skeletal muscle.

Point mutations in beta I sigma 1 spectrin that impair the self-association of spectrin alpha beta heterodimers cause mild to severe hemolytic disease and erythrocyte shape abnormalities. Most such mutations act in a dominant negative fashion. One mutation that is particularly devastating is found in beta spectrin Providence. The Providence mutation replaces serine2019 with proline. Heterozygotes display microcytic and fragile erythrocytes; homozygotes die in the neonatal period. It has recently been determined that an alternative transcript of the same beta I sigma 1 spectrin gene expressed in erythroid lineage cells is the major spectrin in skeletal and cardiac muscle and in some neurons. Because the site of the Providence mutation is common to both beta I sigma 1 and beta I sigma 2 spectrin, defective protein must also be expressed in these tissues. Yet the impact of this or any other beta I spectrin mutation outside of the red cell is unexplored. To address this question with respect to skeletal muscle, we have examined the effects of the Providence mutation in cultured muscle cells, after adoptive gene transfer to adult mice, and in two infants homozygous for spectrin Providence. Transfection of the FLAG epitope tagged wild-type beta I sigma 2 or Providence beta I sigma 2 cDNA constructs into C2C12 myoblasts demonstrated by sedimentation velocity analysis that spectrin beta I sigma 2 Providence formed alpha II/-beta I sigma 2 heterodimers in muscle cells but not heterotetramers. Correspondingly, wild-type beta I sigma 2 spectrin formed both alpha II/beta I sigma 1 dimers and heterotetramers, although the proportion of dimers was surprisingly high, which suggested some limitation on self-association in the muscle environment. After adoptive gene transfer into adult mouse skeletal muscle in vivo, both the wild-type and mutant beta I sigma 2 spectrins assembled into a subsarcolemmal complex in a pattern indistinguishable from the native spectrin skeleton. Skeletal muscle taken at autopsy from two infants homozygous for spectrin Providence was normal histologically, as was the intracellular distribution of beta I sigma 2 spectrin as measured by immunoperoxidase staining. These patients also revealed no clinical evidence of myopathy or muscle wasting. It is unknown if they would have experienced dystrophic or myopathic changes if they had lived longer, although we believe that this is unlikely based on the absence of clinical myopathies in patients with other (albeit less severe) beta I spectrin self-association defects. Collectively, these observations indicate that the spectrin mutations that impact tetramer and oligomer formation, even those with a severe hemolytic phenotype, do not impact skeletal muscle function primarily because skeletal muscle does not use the oligomerizing feature of the spectrin skeleton to the same degree as erythrocytes.

Identification of a small cytoplasmic ankyrin (AnkG119) in the kidney and muscle that binds beta I sigma spectrin and associates with the Golgi apparatus.

Ankyrins are a family of large, membrane-associated proteins that mediate the linkage of the cytoskeleton to a variety of membrane transport and receptor proteins. A repetitive 33-residue motif characteristic of domain I of ankyrin has also been identified in proteins involved with cell cycle control and development. We have cloned and characterized a novel ankyrin isoform, AnkG119 (GenBank accession No. U43965), from the human kidney which lacks part of this repetitive domain and associates in MDCK cells with beta I sigma spectrin and the Golgi apparatus, but not the plasma membrane. Sequence comparison reveals this ankyrin to be an alternative transcript of AnkG, a much larger ankyrin recently cloned from brain. AnkG119 has a predicted size of 119,201 D, and contains a 47-kD domain I consisting of 13 ankyrin repeat units, a 67-kD domain II with a highly conserved spectrin-binding motif, and a truncated 5-kD putative regulatory domain. An AnkG119 cDNA probe hybridized to a 6.0-kb message in human and rat kidney, placenta, and skeletal muscle. An antibody raised to AnkG119 recognized an apparent 116-kD peptide in rat kidney cortical tissue and MDCK cell lysates, and did not react with larger isoforms of ankyrin at 190 and 210 kD in these tissues, nor in bovine brain, nor with ankyrin from human erythrocytes. AnkG119 remains extractable in 0.5% Triton X-100, and assumes a punctuate cytoplasmic distribution in mature MDCK cells, in contrast to the Triton-stable plasma membrane localization of all previously described renal ankyrins. AnkG119 immunocreativity in subconfluent MDCK cells distributes with the Golgi complex in a pattern coincident with beta -COP and beta I sigma spectrin immunoreactivity. A fusion peptide containing residues 669-860 of AnkG119 interacts with beta I sigma 1 spectrin in vitro with a Kd = 4.2 +/- 4.0 ( +/- 2 SD) nM, and avidly binds the beta spectrin in MDCK cell lysates. Collectively, these data identify AnkG119 as a novel small ankyrin that binds and colocalizes with beta I sigma spectrin in the ER and Golgi apparatus, and possible on a subset of endosomes during the early stages of polarity development. We hypothesize that AnkG119 and beta I spectrin form a vesicular Golgi-associated membrane skeleton, promote the organization of protein microdomains within the Golgi and trans-Golgi networks, and contribute to polarized vesicle transport.

Identification of encephalitogenic V beta-4-bearing T cells in SJL mice. Further evidence for the V region disease hypothesis?

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system mediated by T cells bearing TCR of restricted heterogeneity. Thus, in the murine PL strain, V beta-8.2 is used by 80% of the encephalitogenic T cells. This observation has led to the successful prevention and reversal of EAE by the in vivo use of mAb directed to these restricted gene products. In SJL mice, the V beta-17a gene product has been shown to be used by approximately 50% of encephalitogenic T cells subsequent to immunization with a myelin basic protein (MBP)-derived peptide. However, the other V beta genes used by encephalitogenic T cells in SJL EAE have remained uncharacterized. We now report, for the first time, the beta-chain-encoding DNA sequence of two encephalitogenic, MBP-reactive, SJL-derived T cell clones. These clones which are specific for H-2s and the carboxyl-terminus (amino acid 92-103) of MBP, use TCR encoded by V beta-4. In addition, we demonstrate that the transfer of EAE by a heterogenous SJL-derived encephalitogenic T cell line can be prevented using an anti-V beta-4 antibody in vivo. V beta-4 usage has been previously described in a H-2u/MBP amino-terminus-reactive encephalitogenic T cell. The present findings may thus further support the "V region-disease" hypothesis.