Faculty Bibliography

Parathyroid hormone (PTH) regulation of matrix metalloproteinase-13 (MMP-13) expression in osteoblasts contributes to normal bone turnover. The PTH response region of the rat MMP-13 gene spans nucleotides (nt) -148 to -38 and supports binding of numerous transcription factors, including Runx2, necessary for osteoblast differentiation, c-Fos/c-Jun, and Ets-1. These trans-acting proteins mediate hormone induction via incompletely defined combinatorial interactions. Within this region, adjacent to the distal Runx2 site, is a homopolymeric(dA:dT) element (-119/-110 nt) that conforms to the consensus site for the novel transcription factor nuclear matrix protein-4/cas interacting zinc finger protein (Nmp4/CIZ). This protein regulates bone cell expression of type I collagen and suppresses BMP2-enhanced osteoblast differentiation. The aim of this study was to determine whether Nmp4/CIZ contributes to MMP-13 basal transcription and PTH responsiveness in osteoblasts. Electrophoretic mobility shift analysis confirms Nmp4/CIZ binding within the MMP-13 PTH response region. Mutation of the Nmp4/CIZ element decreases basal activity of an MMP-13 promoter-reporter construct containing the first 1329 nt of the 5'-regulatory region, and overexpression of Nmp4/CIZ protein enhances the activity of the wild-type promoter. The same mutation of the homopolymeric(dA:dT) element enhances the MMP-13 response to PTH and PGE(2). Overexpression of Nmp4/CIZ diminishes hormone induction. Mutation of both the homopolymeric(dA:dT) element and the adjacent Runx2 site further augments the PTH response. On the basis of these data and previous studies, we propose that Nmp4/CIZ is a component of a multiprotein assemblage or enhanceosome within the MMP-13 PTH response region and that, within this context, Nmp4/CIZ promotes both basal expression and hormonal synergy

AIM/OBJECTIVE:To study the effect of injecting hematopoietic stem cells containing the preproinsulin gene II (rI2) via recombinant adeno-associated virus (rAAV) into normal and streptozotocin-diabetic rats. METHODS:rI2 was transfected into rat hematopoietic stem cells using rAAV vector. Stem cells were injected by intravenous route into normal and STZ-induced diabetic rats to study blood sugar and expression of rI2 in various tissues. The pLP-1 recombinant plasmid containing rI2 (vLP-1) was engineered as previously described. Bone marrow from female Wistar-Furth rats was enriched for stem cells by using plastic adherence and monoclonal antirat CD3 and CD45 RA to deplete T and B cells. The remaining cells were exposed to vLP-1 (multiplicity of infection MOI =50:1 or 100:1) for 2 h. Approximately ten million exposed stem cells were injected by intravenous route into each animal; there were four groups: normal animals at MOI 50:1 (group 1) or MOI 100:1 (group 2); group 3 animals (n = 9) were streptozotocin-induced diabetic animals at MOI 100:1. Animals that showed reversal of diabetes from group 3 were sacrificed for study of gene expression at weeks 1, 2, and 6, respectively. Control diabetic animals did not receive stem cells or virus constituted group 4. Expression of rI2 was analyzed by RT-PCR and Southern analyses. RESULTS:Despite introduction of insulin gene, groups 1 and 2 had blood sugar concentrations that remained within normal levels, while 3 of 9 animals in group 3 showed reversal of diabetes; using RT-PCR,group 1 expressed rI2 in liver, spleen, thymus, brain, and heart at week 1 only. In group 2, rI2 was seen in the thymus up to 6 weeks; in diabetic animals (group 3) rI2 was seen in liver, bone marrow, spleen, thymus, and peripheral blood lymphocytes at week 2 and in thymus and lymphocytes at week 6. CONCLUSIONS:We have shown that (1) rAAV is a useful vector for transferring rI2 into rat hematopoietic stem cells; (2) normal animals remained euglycemic after injection of stem cells containing rI2 despite identification in various tissues suggesting autoregulation, and (3) short-term reversal of diabetes was achieved in some animals by injection of stem cells containing rI2.

Splice variants of the Nmp4 gene include nuclear matrix transcription factors that regulate the type I collagen alpha1(I) polypeptide chain (COL1A1) promoter and several matrix metalloproteinase (MMP) genes. To date, these are the only Cys(2)His(2) zinc finger proteins known to bind within the minor groove of homopolymeric (dA.dT) DNA. Nmp4 isoforms contain from 5 to 8 Cys(2)His(2) zinc fingers, an SH3-binding domain that overlaps with a putative AT-hook and a polyglutamine-alanine repeat (poly(QA)). To determine the mechanistic significance of Cys(2)His(2) zinc finger association with this unusual consensus DNA binding element, we identified the Nmp4 DNA-binding and transcriptional activation domains. Zinc fingers 2, 3, and 6 mediated association with the homopolymeric (dA.dT) COL1A1/MMP DNA consensus element. The N terminus of the Nmp4 protein exhibited a strong trans-activation capacity when fused to the GAL4 DNA-binding domain, but this activity was masked within the context of the full-length Nmp4-GAL4 DNA-binding domain chimera. However, upon binding to the COL1A1/MMP homopolymeric (dA.dT) element, the native Nmp4 protein up-regulated transcription, and the poly(QA) domain acquired a significant role in trans-activation. We propose that allosteric effects induced upon zinc finger association with the homopolymeric (dA.dT) minor groove confer context-specific functionality to this unusual family of Cys(2)His(2) transcription factors.