Mari K. Davidson

Protocadherins: a large family of cadherin-related molecules in central nervous system.

Using the polymerase chain reaction, we have isolated numerous rat and human cDNAs of which the deduced amino acid sequences are highly homologous to the sequences of the extracellular domain of cadherins. The entire putative coding sequences for two human proteins defined by two of these cDNAs have been determined. The overall structure of these molecules is very similar to that of classic cadherins, but they have some unique features. The extracellular domains are composed of six or seven subdomains that are very similar to those of cadherins, but have characteristic properties. The cytoplasmic domains, on the other hand, have no significant homology with those of classic cadherins. Since various cDNAs with almost identical features were obtained also from Xenopus, Drosophila and Caenorhabditis elegans, it appears that similar molecules are expressed in a variety of organisms. We have tentatively named these proteins protocadherins. They are highly expressed in brain and their expression appears to be developmentally regulated. The proteins expressed from the two full‐length cDNAs in L cells were approximately 170 or 150 kDa in size, and were localized mainly at cell‐cell contact sites. Moreover, the transfectants showed cell adhesion activity.

Human Osteoblasts Express a Repertoire of Cadherins, Which Are Critical for BMP‐2–Induced Osteogenic Differentiation

Direct cell–cell interactions are fundamental for tissue development and differentiation. We have studied the expression and function of cadherins in human osteoblasts during in vitro differentiation. Using reverse transcription‐polymerase chain reaction and mRNA hybridization, we found that human trabecular bone osteoblasts (HOBs), osteoprogenitor marrow stromal cells (BMCs), and the osteogenic sarcoma lines, SaOS‐2 and MG‐63, expressed mRNA for cadherin‐11 (C11) and N‐cadherin (N‐cad). HOBs and BMCs also expressed low levels of cadherin‐4 (C4) mRNA. C11 was the most abundant cadherin protein present in human osteoblasts, and its expression was unaffected by bone morphogenetic protein‐2 (BMP‐2) treatment of either BMCs or HOBs. Likewise, N‐cad mRNA did not change during BMP‐2 incubation. Conversely, C4 protein, undetectable in transformed cell lines, was down‐regulated by BMP‐2 treatment of normal cells. Both C11 and C4 were localized to sites of cell–cell contact in both HOBs and BMCs, colocalized with β‐catenin, and bands corresponding to cadherins were coimmunoprecipitated by a β‐catenin antibody, findings indicative of functional cadherins. A decapeptide containing the HAV motif of human N‐cad partially inhibited Ca2+‐dependent cell–cell adhesion and completely prevented BMP‐2–induced stimulation of alkaline phosphatase activity by BMCs. Thus, human osteoblasts and their progenitor cells express a repertoire of multiple cadherins. Cadherin‐mediated cell‐to‐cell adhesion is critical for normal human osteoblast differentiation.

Boosting epidermal growth factor receptor expression by gene gun transfection stimulates epidermal growth in vivo

Expression constructs encoding a full‐length cDNA encoding the human epidermal growth factor receptor, or reporter gene for green fluorescent protein or luciferase were coated onto gold particles and driven into porcine skin using a gene gun delivery system. Strategies for epidermal growth factor receptor boosting were tested in two types of wounds. For grafted wounds, intact porcine skin was pretreated by the introduction of the epidermal growth factor receptor expression construct 24 hours before its harvesting as a split‐thickness skin graft. Partial‐thickness excisional wound beds (donor sites) were transfected at the time of their creation. Wound healing parameters were subsequently tested in the presence or absence of excess epidermal growth factor ligand. Initial distributions of gene gun delivered gold particles as well as luciferase expression levels suggested that optimal skin penetrations and expression levels were achieved at 500 psi for intact epidermis and 300 psi for exposed wound beds. At 2 days after gene delivery, visualization of green fluorescent protein by fluorescence microscopy showed focal expression of green fluorescent protein at the advancing epithelial outgrowths found at wound edges or surviving epithelial remnants. Green fluorescent protein expression appeared transient since no green fluorescent protein was noted in specimens removed at 4 days after injury. Northern blot analysis on mRNA isolated from wounds 2 days after introduction of epidermal growth factor receptor coated gold particles by gene gun confirmed the expression of the human epidermal growth factor receptor transgene in both skin grafts and excisional wounds. Skin grafts showed subsequent biological responses to the introduction of excessive epidermal growth factor receptor as well as expression of the human epidermal growth factor receptor construct within healing epidermis. While control autografts (reporter gene treated, epidermal growth factor alone, placebo formula, no treatment) showed few 5′‐bromodeoxyuridine‐labeled cells, epidermal growth factor receptor autografts showed 5′‐bromodeoxyuridine labeling of nearly every basal cell. Favorable wound healing outcomes were also shown within excisional wounds following in vivo boosting of epidermal growth factor receptor. Four days after receiving epidermal growth factor receptor particle bombardment, resurfacing was significantly accelerated in those wounds receiving epidermal growth factor receptor transgene. Application of topical epidermal growth factor ligand resulted in the highest percentage of resurfacing. Maximal re‐epithelialization was noted in wound beds receiving both receptor boosting and excessive daily epidermal growth factor ligand. A modest increase in the thickness of the granulation tissue followed gene therapy with epidermal growth factor receptor. In summary these in vivo data suggest that it is possible to boost in vivo expression of a tyrosine kinase receptor during wound repair. Increased epidermal growth factor receptor expression has an integral impact on cell proliferation, rates of resurfacing and dermal components and merits consideration as a possible therapeutic agent.

Distinct functions of S. pombe Rec12 (Spo11) protein and Rec12-dependent crossover recombination (chiasmata) in meiosis I; and a requirement for Rec12 in meiosis II

BackgroundIn most organisms proper reductional chromosome segregation during meiosis I is strongly correlated with the presence of crossover recombination structures (chiasmata); recombination deficient mutants lack crossovers and suffer meiosis I nondisjunction. We report that these functions are separable in the fission yeast Schizosaccharomyces pombe.ResultsIntron mapping and expression studies confirmed that Rec12 is a member of the Spo11/Top6A topoisomerase family required for the formation of meiotic dsDNA breaks and recombination. rec12-117, rec12-D15 (null), and rec12-Y98F (active site) mutants lacked most crossover recombination and chromosomes segregated abnormally to generate aneuploid meiotic products. Since S. pombe contains only three chromosome pairs, many of those aneuploid products were viable. The types of aberrant chromosome segregation were inferred from the inheritance patterns of centromere linked markers in diploid meiotic products. The rec12-117 and rec12-D15 mutants manifest segregation errors during both meiosis I and meiosis II. Remarkably, the rec12-Y98F (active site) mutant exhibited essentially normal meiosis I segregation patterns, but still exhibited meiosis II segregation errors.ConclusionsRec12 is a 345 amino acid protein required for most crossover recombination and for chiasmatic segregation of chromosomes during meiosis I. Rec12 also participates in a backup distributive (achiasmatic) system of chromosome segregation during meiosis I. In addition, catalytically-active Rec12 mediates some signal that is required for faithful equational segregation of chromosomes during meiosis II.

Epiregulin is more potent than EGF or TGFα in promoting in vitro wound closure due to enhanced ERK/MAPK activation

Epiregulin (EPR) is a broad specificity EGF family member that activates ErbB1 and ErbB4 homodimers and all possible heterodimeric ErbB complexes. We have previously shown that topical EPR enhances the repair of murine excisional wounds. The purpose of this study was to determine whether EPR was more effective than EGF or TGFα in promoting in vitro wound closure and to compare the EPR induced signal transduction pathways with those activated by EGF and TGFα. Normal human epidermal keratinocytes or A431 cells were scratch wounded and treated for 24 h with varying doses of EPR, EGF or TGFα. Five‐fold lower doses of EPR were significantly better than EGF or TGFα in stimulating in vitro wound closure. Mitomycin‐c reduced EPR induced wound closure by 59%, versus a 9% and 25% decrease in EGF and TGFα induced closure. The ERK/MAPK inhibitor PD‐98059 decreased EPR induced wound closure by 88%. By contrast, the PLC inhibitor U‐73122, only reduced the EPR induced response by 21%. Immunoblot analysis revealed that 2 nM EPR stimulated a six‐fold increase in p‐ERK1/2, whereas 10 nM EGF or TGFα stimulated only a 3‐ and 2.5‐fold increase in p‐ERK1/2. When compared with EGF or TGFα, EPR is a more potent and more effective inducer of in vitro wound closure due to its ability to promote significantly greater ERK/MAPK activation.

Atf1-Pcr1-M26 complex links stress-activated MAPK and cAMP-dependent protein kinase pathways via chromatin remodeling of cgs2+.

Although co-ordinate interaction between different signal transduction pathways is essential for developmental decisions, interpathway connections are often obscured and difficult to identify due to cross-talk. Here signals from the fission yeast stress-activated MAPK Spc1 are shown to regulate Cgs2, a negative regulator of the cAMP-dependent protein kinase (protein kinase A) pathway. Pathway integration is achieved via Spc1-dependent binding of Atf1-Pcr1 heterodimer to an M26 DNA site in the cgs2+ promoter, which remodels chromatin to regulate expression of cgs2+ and targets downstream of protein kinase A. This direct interpathway connection co-ordinates signals of nitrogen and carbon source depletion to affect a G0 cell-cycle checkpoint and sexual differentiation. The Atf1-Pcr1-M26 complex-dependent chromatin remodeling provides a molecular mechanism whereby Atf1-Pcr1 heterodimer can function differentially as either a transcriptional activator, or as a transcriptional repressor, or as an inducer of meiotic recombination. We also show that the Atf1-Pcr1-M26 complex functions as both an inducer and repressor of chromatin remodeling, which provides a way for various chromatin remodeling-dependent effector functions to be regulated.

Differential regulation of cadherins by dexamethasone in human osteoblastic cells.

Human osteoblasts express a repertoire of cadherins, including N‐cadherin (N‐cad), cadherin‐11 (C11), and cadherin‐4 (C4). We have previously shown that direct cell‐cell adhesion via cadherins is critical for BMP‐2‐induced osteoblast differentiation. In this study, we have analyzed the regulation of cadherin expression in normal human trabecular bone osteoblasts (HOB), and osteoprogenitor marrow stromal cells (BMC), during exposure to dexamethasone, another inducer of human bone cell differentiation. Dexamethasone inhibited the expression of both C11 and N‐cad mRNA in both BMC and HOB, although the effect was much more pronounced on N‐cad than on C11. This action of the steroid was dose dependent, was maximal at 10−7 M concentration, and occurred as early as after 1 day of incubation. By contrast, expression of C4 mRNA and protein was strongly induced by dexamethasone in BMC and was stimulated in HOB. This stimulatory effect lasted for at least 2 weeks of incubation. A cadherin inhibitor, HAV‐containing decapeptide only partially (∼50%) prevented dexamethasone‐induced stimulation of alkaline phosphatase activity by BMC, which instead was not altered by incubation with a neutralizing antibody against C4. Therefore, the pattern of cadherin regulation by dexamethasone radically differs form that observed with BMP‐2. Dexamethasone effects on certain osteoblast differentiated features, such as induction of alkaline phosphatase activity are not strictly dependent on cadherin function. J. Cell. Biochem.77:499–506, 2000.

Discrete DNA sites regulate global distribution of meiotic recombination

Homologous recombination is induced to high levels in meiosis, is initiated by Spo11-catalyzed DNA double-strand breaks (DSBs) and is clustered at hotspots that regulate its positioning in the genome. Recombination is required for proper chromosome segregation in meiosis and defects in its frequency or positioning cause chromosome mis-segregation and, consequently, congenital birth defects such as Downs syndrome. Therefore, elucidating how meiotic recombination is positioned is of fundamental and biomedical interest. Our integration of historical and contemporary advances in the field, plus the re-analysis of published microarray data on the genome-wide distribution of recombination supports a unifying model for such regulation. We posit that discrete DNA sequence motifs position and regulate essentially all recombination across the genome, in much the same way that DNA sites position and regulate transcription. Moreover, we illustrate the use of overlapping mechanisms for the regulation of transcription and meiotic recombination. Bound transcription factors induce histone modifications that position recombination at hotspots.

Donor Origin of Circulating Endothelial Progenitors After Allogeneic Bone Marrow Transplantation

Endothelial cell precursors circulate in blood and express antigens found on hematopoietic stem cells, suggesting that such precursors might be subject to transplantation. To investigate, we obtained adherence-depleted peripheral blood mononuclear cells from 3 individuals who had received a sex-mismatched allogeneic bone marrow transplant (BMT) and cultured the cells on fibronectin-coated plates with endothelial growth factors. The phenotype of the spindle-shaped cells that emerged in culture was characterized by immunofluorescent staining, and the origin of the cells was determined using a polymerase chain reaction (PCR)-based assay for polymorphic short tandem repeats (STRs). The cells manifested a number of endothelial characteristics-such as von Wlllebrand factor, CD31, and Flk-1/KDR expression; Bandeiraea simplicifolia lectin 1 binding; and acetylated low-density lipoprotein uptake-but lacked expression of certain markers of activation or differentiation, including intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and the epitope for the anti-endothelial cell antibody P1H12. For each patient and at all time points studied (ranging from 5 to 52 months after transplantation), STR-PCR analysis showed that cultured cells and nucleated blood cells came exclusively from the bone marrow donor. These results demonstrate that circulating endothelial progenitors are both transplantable and capable of long-term repopulation of human allogeneic BMT recipients.

Meiotic Recombination Hotspots of Fission Yeast Are Directed to Loci that Express Non-Coding RNA

Background Polyadenylated, mRNA-like transcripts with no coding potential are abundant in eukaryotes, but the functions of these long non-coding RNAs (ncRNAs) are enigmatic. In meiosis, Rec12 (Spo11) catalyzes the formation of dsDNA breaks (DSBs) that initiate homologous recombination. Most meiotic recombination is positioned at hotspots, but knowledge of the mechanisms is nebulous. In the fission yeast genome DSBs are located within 194 prominent peaks separated on average by 65-kbp intervals of DNA that are largely free of DSBs. Methodology/Principal Findings We compared the genome-wide distribution of DSB peaks to that of polyadenylated ncRNA molecules of the prl class. DSB peaks map to ncRNA loci that may be situated within ORFs, near the boundaries of ORFs and intergenic regions, or most often within intergenic regions. Unconditional statistical tests revealed that this colocalization is non-random and robust (P≤5.5×10−8). Furthermore, we tested and rejected the hypothesis that the ncRNA loci and DSB peaks localize preferentially, but independently, to a third entity on the chromosomes. Conclusions/Significance Meiotic DSB hotspots are directed to loci that express polyadenylated ncRNAs. This reveals an unexpected, possibly unitary mechanism for what directs meiotic recombination to hotspots. It also reveals a likely biological function for enigmatic ncRNAs. We propose specific mechanisms by which ncRNA molecules, or some aspect of RNA metabolism associated with ncRNA loci, help to position recombination protein complexes at DSB hotspots within chromosomes.