Citations

Proteolytic cleavage of the six known insulin-like growth factor binding proteins (IGFBPs) is a powerful means of rapid structure and function modification of these important growth-regulatory proteins. Intact IGFBP-4 is a potent inhibitor of IGF action in vitro, and cleavage of IGFBP-4 has been shown to abolish its ability to inhibit IGF stimulatory effects in a variety of systems, suggesting that IGFBP-4 proteolysis acts as a positive regulator of IGF bioavailability. Here we report the isolation of an IGF-dependent IGFBP-4-specific protease from human fibroblast-conditioned media and its identification by mass spectrometry microsequencing as pregnancy-associated plasma protein-A (PAPP-A), a protein of unknown function found in high concentrations in the maternal circulation during pregnancy. Antibodies raised against PAPP-A both inhibited and immunodepleted IGFBP-4 protease activity in human fibroblast-conditioned media. Moreover, PAPP-A purified from pregnancy sera had IGF-dependent IGFBP-4 protease activity. PAPP-A mRNA was expressed by the human fibroblasts and osteoblasts, and PAPP-A protein was secreted into the culture medium. In conclusion, we have identified an IGF-dependent IGFBP protease and at the same time assigned a function to PAPP-A. This represents an unanticipated union of two areas of research that were not linked in any way before this report.

Translocations involving c -myc and an Ig locus have been reported rarely in human multiple myeloma (MM). Using specific fluorescence in situ hybridization probes, we show complex karyotypic abnormalities of the c -myc or L -myc locus in 19 of 20 MM cell lines and approximately 50% of advanced primary MM tumors. These abnormalities include unusual and complex translocations and insertions that often juxtapose myc with an IgH or IgL locus. For two advanced primary MM tumors, some tumor cells contain a karyotypic abnormality of the c -myc locus, whereas other tumor cells do not, indicating that this karyotypic abnormality of c -myc occurs as a late event. All informative MM cell lines show monoallelic expression of c -myc . For Burkitt's lymphoma and mouse plasmacytoma tumors, balanced translocation that juxtaposes c -myc with one of the Ig loci is an early, invariant event that is mediated by B cell-specific DNA modification mechanisms. By contrast, for MM, dysregulation of c -myc apparently is caused principally by complex genomic rearrangements that occur during late stages of MM progression and do not involve B cell-specific DNA modification mechanisms.

Presenilins mediate an unusual intramembranous proteolytic activity known as γ-secretase, two substrates of which are the Notch receptor (Notch) and the β-amyloid precursor protein (APP). γ-Secretase-mediated cleavage of APP, like that of Notch, yields an intracellular fragment [ A PP i ntra c ellular d omain (AICD)] that forms a transcriptively active complex. We now demonstrate a functional role for AICD in regulating phosphoinositide-mediated calcium signaling. Genetic ablation of the presenilins or pharmacological inhibition of γ-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner through a mechanism involving the modulation of endoplasmic reticulum calcium stores. Cells lacking APP (and hence AICD) exhibited similar calcium signaling deficits, and—notably—these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Our findings indicate that the AICD regulates phosphoinositide-mediated calcium signaling through a γ-secretase-dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analogous to that of Notch.

Cyclic ADP ribose (cADPR) has been shown to trigger Ca 2+ release from intracellular stores through ryanodine receptor/channel. In our previous study we observed that all- trans- retinoic acid stimulates cADPR synthesis by ADP ribose cyclase (ADPR cyclase) in cultured epithelial cells. We have now investigated whether cADPR may play a signaling role in action of β-estradiol (E 2 ), an archetypal steroid superfamily hormone, upon its major target organ, uterus, in vivo . Administration of E 2 to gonadectomized rats (0.2 mg/kg per day for 7 days) resulted in an ≈Δ + 300% increase of ADPR cyclase activity in extracts from uterus, but in liver, brain, or skeletal muscle ADPR cyclase was unchanged. Most of the E 2 -stimulated uterine ADPR cyclase was associated with membranes. The higher ADPR cyclase activity in response to E 2 was due to the increase of V MAX without change in K m . Simultaneous administration of estrogen antagonist tamoxifen (8 mg/kg per day) with E 2 (0.2 mg/kg per day) prevented an increase in ADPR cyclase. In uterine extracts from E 2 -treated rats, the rate of cADPR inactivation by cADPR hydrolase and the activity of NADase was increased, but to a much lesser degree than activity of ADPR cyclase. Our results indicate that E 2 , via action to its nuclear receptors in vivo , increases ADPR cyclase activity in uterus. We propose that some of the estrogen effects, and by extension the effects of other steroid superfamily hormones, upon specialized cellular functions and upon hormone-induced gene expression in target cells, are mediated by cADPR-Ca 2+ release pathway.

CD1d-restricted invariant NKT (iNKT) cells are immunoregulatory cells whose loss exacerbates diabetes in nonobese diabetic (NOD) female mice. Here, we show that the relative numbers of iNKT cells from the pancreatic islets of NOD mice decrease at the time of conversion from peri-insulitis to invasive insulitis and diabetes. Conversely, NOD male mice who have a low incidence of diabetes showed an increased frequency of iNKT cells. Moreover, administration of α-galactosylceramide, a potent activating ligand presented by CD1d, ameliorated the development of diabetes in NOD female mice and resulted in the accumulation of iNKT cells and myeloid dendritic cells (DC) in pancreatic lymph nodes (PLN), but not in inguinal lymph nodes. Strikingly, injection of NOD female mice with myeloid DC isolated from the PLN, but not those from the inguinal lymph nodes, completely prevented diabetes. Thus, the immunoregulatory role of iNKT cells is manifested by the recruitment of tolerogenic myeloid DC to the PLN and the inhibition of ongoing autoimmune inflammation.

The avian erythroblastosis viral oncogene (v- erb B) encodes a receptor tyrosine kinase that possesses sarcomagenic and leukemogenic potential. We have expressed transforming and nontransforming mutants of v- erb B in fibroblasts to detect transformation-associated signal transduction events. Coimmunoprecipitation and affinity chromatography have been used to identify a transformation-associated, tyrosine phosphorylated, multiprotein complex. This complex consists of Src homologous collagen protein (Shc), growth factor receptor binding protein 2 (Grb2), son of sevenless (Sos), and a novel tyrosine phosphorylated form of the cytoskeletal regulatory protein caldesmon. Immunofluorescence localization studies further reveal that, in contrast to the distribution of caldesmon along actin stress fibers in normal fibroblasts, caldesmon colocalizes with Shc in plasma membrane blebs in transformed fibroblasts. This colocalization of caldesmon and Shc correlates with actin stress fiber disassembly and v- erb B-mediated transformation. The tyrosine phosphorylation of caldesmon, and its association with the Shc–Grb2–Sos signaling complex directly links tyrosine kinase oncogenic signaling events with cytoskeletal regulatory processes, and may define one mechanism regulating actin stress fiber disassembly in transformed cells.