Hemopoietic cells have been reported to synthesize insulin-like growth factor-I (IGF-I) messenger RNA (mRNA), but the relative contribution of specific cell lineages that express these transcripts remains unknown. Reverse transcription and amplification of complementary DNA (cDNA) by the polymerase chain reaction were used to characterize full-length IGF-I mRNA transcripts in murine hemopoietic cells. The identity of transcripts encoding the entire prepropeptide was confirmed by restriction endonuclease digestion, Southern blotting, cloning, and Sanger sequencing. Abundance of IGF-I mRNA transcripts was assessed both by Northern blotting and sensitive ribonuclease protection assays followed by quantification with Phosphor-Imager analysis. Whereas IGF-I cDNA transcripts could be detected in a variety of leukocytes after polymerase chain reaction amplification, IGF-I mRNA was negligible or nondetectable in T and B cell lines and in those tissues containing a predominance of these cell types (e.g. spleen and thymus) by Northern blotting and ribonuclease protection assays. In contrast, elicited peritoneal macrophages, a macrophage cell line, microglia, and bone marrow macrophages differentiated in vitro expressed abundant IGF-I mRNA transcripts, whereas neither a premyeloid cell line nor freshly isolated bone marrow cells expressed significant transcripts. The 5'-identity of macrophage IGF-I transcripts was established using an exon 2-derived IGF-I cDNA probe. All protected transcripts were foreshortened, indicating transcript initiation exclusively within exon 1, characteristic of extra-hepatic IGF-I mRNA. However, at the 3'-end, both IGF-I Ea (lacking exon 5) and IGF-I Eb (containing exon 5) mRNA transcripts were evident, with the Eb product being detected at levels similar to those present in hepatic cellular RNA. A large molecular size (26 kilodaltons) prepro-IGF-I peptide was also detected in macrophage cell lysates by Western blotting. Collectively, our observations show that: 1) among hemopoietic cells, myeloid rather than lymphoid cells are the major source of IGF-I; 2) macrophage IGF-I mRNA consists of class I Ea and Eb transcripts; 3) these transcripts are translated into protein; and 4) expression of IGF-I is directly associated with differentiation of bone marrow macrophages.

Conventional ankyrins are cortical cytoskeletal proteins that form an ankyrin-spectrin meshwork underlying the plasma membrane. We report here the unusual structure of a novel ankyrin (AO13 ankyrin, 775,369 Da, 6994 aa, pI = 4.45) that is required for proper axonal guidance in Caenorhabditis elegans. AO13 ankyrin contains the ANK repeat and spectrin-binding domains found in other ankyrins, but differs from all others in that the acidic carboxyl region contains six blocks of serine/threonine/glutamic acid/proline rich (STEP) repeats separated by seven hydrophobic domains. The STEP repeat blocks are composed primarily of sequences related to ETTTTTTVTREHFEPED(E/D)X(n)VVESEEYSASGSPVPSE (E/K)DVE(H/R)VI, and the hydrophobic domains contain sequences related to PESGEESDGEGFGSKVLGFAKK[AGMVAGGVVAAPVALAAVGA]KAAYDALKKDDDEE, which includes a potential transmembrane domain (in brackets). Recombinant protein fragments of AO13 ankyrin were used to prepare polyclonal antisera against the spectrin-binding domain (AO271 Ab), the conventional ankyrin regulatory domain (AO280 Ab), the AO13 ankyrin STEP domain (AO346 Ab), the AO13 ankyrin STEP + hydrophobic domain (AO289 Ab), and against two carboxyl terminal domain fragments (AO263 Ab and AO327 Ab). Western blot analysis with these Ab probes demonstrated multiple protein isoforms. By immunofluorescence microscopy, the antispectrin-binding and regulatory domain (AO271 and AO280) antibodies recognized many cell types, including neurons, and stained the junctions between cells. The AO13 ankyrin-specific (AO289 and AO346) antibodies showed a neurally restricted pattern, staining nerve processes and the periphery of neural cell bodies. These results are consistent with a role for AO13 ankyrin in neural development.

Spontaneous production of insulin-like growth factor-I (IGF-I) by inflammatory macrophages contributes to aberrant wound healing, but little is known about regulation of IGF-I synthesis in myeloid cells. The T cell-derived cytokine interferon-gamma (IFN gamma) inhibits several fibrogenic and angiogenic components of the wound-healing response. We have used metabolic labeling of primary colony stimulating factor-1 (CSF-1)-derived macrophages and a transformed macrophage cell line (PU5-1R) followed by immunoprecipitation to demonstrate that synthesis of the 17 kilodalton (kDa) prepro-IGF-I protein by these cells is substantially inhibited by IFN gamma. An exon 4 IGF-I/beta-actin riboprobe expression cassette was used in RNase protection assays to show that IFN gamma also reduces steady state levels of IGF-I mRNA in three different populations of macrophages in a time- and dose-dependent manner. This effect is specific for IFN gamma because neither the IFNs-alpha/beta nor lipopolysaccharide (LPS) affects expression of steady state IGF-I transcripts. Down-regulation of IGF-I mRNA by IFN gamma is dependent on de novo protein synthesis and is abrogated by coculture with cycloheximide. Nuclear run-on assays revealed that elongation of IGF-I transcripts is absent in fresh bone marrow cells but is induced several-fold after cells are cultured for 6 days with CSF-1. Treatment of these CSF-1-derived macrophages with IFN gamma for 6 h substantially inhibits synthesis of IGF-I mRNA. Studies on the decay of IGF-I mRNA in PU5-1R macrophages treated with an RNA polymerase inhibitor confirmed that the decline in IGF-I steady state mRNA in IFN gamma-treated cultures arises from an inhibition of transcription rather than from a reduction in mRNA stability. Since a variety of inflammatory mediators can induce expression of IGF-I in macrophages, inhibition of macrophage IGF-I synthesis by IFN gamma provides a mechanism by which leukocytes regulate levels of this growth factor in their microenvironment.

Rapidly proliferating transformed mammalian cells can be photodestroyed in vitro upon inducing the accumulation of endogenous protoporphyrin IX (Proto). Proto biosynthesis and accumulation were triggered by manipulation of the porphyrin-heme biosynthetic pathway. Proto accumulation in cultured cells was induced by treatment with 1.0 mM delta-aminolevulinic acid (ALA), a naturally occurring 5-carbon amino acid, for 3.5 h. In darkness, significant Proto accumulation became evident within 3.5 h of incubation. In the light, the accumulated tetrapyrroles triggered destruction of treated cells within the first 30 min of illumination, probably via the rapid oxidation of cellular constituents by singlet oxygen. Protoporphyrin IX accumulation and specific cell lysis increased significantly by inclusion of 0.75 mM 1,10-phenanthroline (Oph), a tetrapyrrole biosynthesis modulator. Slower growing untransformed cells did not accumulate significant amounts of Proto following ALA and Oph treatment unless stimulated to proliferate with the mitogenic lectin Concanavalin A.

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