The host response to tissue injury requires a complex interplay of diverse cellular, humoral, and connective tissue elements. Fibroblasts participate in this process by proliferating within injured sites and contributing to scar formation and the long-term remodeling of damaged tissue. Fibroblasts present in areas of tissue injury generally have been regarded to arise by recruitment from surrounding connective tissue; however this may not be the only source of these cells. Long-term culture of adherent, human, and murine leukocyte subpopulations was combined with a variety of immunofluorescence and functional analyses to identify a blood-borne cell type with fibroblast-like properties. We describe for the first time a population of circulating cells with fibroblast properties that specifically enter sites of tissue injury. This novel cell type, termed a “fibrocyte,” was characterized by its distinctive phenotype (collagen+/vimentin+/CD34+), by its rapid entry from blood into subcutaneously implanted wound chambers, and by its presence in connective tissue scars. Blood-borne fibrocytes contribute to scar formation and may play an important role both in normal wound repair and in pathological fibrotic responses.

/pdf/circulating-fibrocytes-define-a-new-leukocyte-subpopulation-1puo5dsowk.pdf

Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair.

cDNA clones encoding three classes of human actins have been isolated and characterized. The first two classes (gamma and beta, cytoplasmic actins) were obtained from a cDNA library constructed from simian virus 40-transformed human fibroblast mRNA, and the third class (alpha, muscle actin) was obtained from a cDNA library constructed from adult human muscle mRNA. A new approach was developed to enrich for full-length cDNAs. The human fibroblast cDNA plasmid library was linearized with restriction enzymes that did not cut the inserts of interest; it was then size-fractionated on gels, and the chimeric molecules of optimal length were selected for retransformation of bacteria. When the resulting clones were screened for actin-coding sequences it was found that some full-length cDNAs were enriched as much as 50- to 100-fold relative to the original frequency of full-length clones in the total library. Two types of clones were distinguished. One of these clones encodes gamma actin and contains 100 base pairs of 5' untranslated region, the entire protein coding region, and the 3' untranslated region. The second class encodes beta actin, and the longest such clone contains 45 base pairs of 5' untranslated region plus the remainder of the mRNA extending to the polyadenylic acid tail. A third class, obtained from the human muscle cDNA library, encodes alpha actin and contains 100 base pairs of 5' untranslated region, the entire coding region, and the 3' untranslated region. Analysis of the DNA sequences of the 5' end of the clones demonstrated that although beta- and gamma-actin genes start with a methionine codon (MET-Asp-Asp-Asp and MET-Glu-Glu-Glu, respectively), the alpha-actin gene starts with a methionine codon followed by a cysteine codon (MET-CYS-Asp-Glu-Asp-Glu). Since no known actin proteins start with a cysteine, it is likely that post-translational removal of cysteine in addition to methionine accompanies alpha-actin synthesis but not beta- and gamma-actin synthesis. This observation has interesting implications both for actin function and actin gene regulation and evolution.

Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed.

The presence of macrophages is required for the regeneration of many cell types during wound healing. Macrophages have been reported to express a wide range of mitogenic factors and cytokines, but none of these factors has been shown in vivo to sustain all the wound-healing processes. It has been suggested that transforming growth factor-alpha (TGF-alpha) may mediate angiogenesis, epidermal regrowth, and formation of granulation tissue in vivo. Macrophages isolated from a wound site, and not exposed to cell culture conditions, expressed messenger RNA transcripts for TGF-alpha, TGF-beta, platelet-derived growth factor A-chain, and insulin-like growth factor-1. The expression of these transcripts was determined by a novel method for RNA analysis in which low numbers of mouse macrophages were isolated from wound cylinders, their RNA was purified and reverse-transcribed, and the complementary DNA was amplified in a polymerase chain reaction primed with growth factor sequence-specific primers. This single-cell RNA phenotyping procedure is rapid and has the potential for quantification, and mRNA transcripts from a single cell or a few cells can be unambiguously demonstrated, with the simultaneous analysis of several mRNA species. Macrophages from wounds expressed TGF-alpha antigen, and wound fluids contained TGF-alpha. Elicited macrophages in culture also expressed TGF-alpha transcripts and polypeptide in a time-dependent manner after stimulation with modified low-density lipoproteins and lipopolysaccharide endotoxin, which are characteristic of the activators found in injured tissues.

https://science.sciencemag.org/content/sci/241/4866/708.full.pdf

Wound Macrophages Express TGF-α and Other Growth Factors in Vivo: Analysis by mRNA Phenotyping

A broad spectrum of biological activities has been proposed for transforming growth factor-beta 3 (TGF-beta 3). To study TGF-beta 3 function in development, TGF-beta 3 null mutant mice were generated by gene-targeting. Within 20 hours of birth, homozygous TGF-beta 3-/- mice die with unique and consistent phenotypic features including delayed pulmonary development and defective palatogenesis. Unlike other null mutants with cleft palate, TGF-beta 3-/- mice lack other concomitant craniofacial abnormalities. This study demonstrates an essential function for TGF-beta 3 in the normal morphogenesis of palate and lung, and directly implicates this cytokine in mechanisms of epithelial-mesenchymal interaction.

Abnormal lung development and cleft palate in mice lacking TGF-beta 3 indicates defects of epithelial-mesenchymal interaction.

Hepcidin is a key regulator of systemic iron homeostasis. Hepcidin deficiency induces iron overload, whereas hepcidin excess induces anemia. Mutations in the gene encoding hemojuvelin (HFE2, also known as HJV) cause severe iron overload and correlate with low hepcidin levels, suggesting that hemojuvelin positively regulates hepcidin expression. Hemojuvelin is a member of the repulsive guidance molecule (RGM) family, which also includes the bone morphogenetic protein (BMP) coreceptors RGMA and DRAGON (RGMB). Here, we report that hemojuvelin is a BMP coreceptor and that hemojuvelin mutants associated with hemochromatosis have impaired BMP signaling ability. Furthermore, BMP upregulates hepatocyte hepcidin expression, a process enhanced by hemojuvelin and blunted in Hfe2-/- hepatocytes. Our data suggest a mechanism by which HFE2 mutations cause hemochromatosis: hemojuvelin dysfunction decreases BMP signaling, thereby lowering hepcidin expression.

Bone morphogenetic protein signaling by hemojuvelin regulates hepcidin expression.

We have determined the sequences of three recombinant cDNAs complementary to different mouse actin mRNAs that contain more than 90% of the coding sequences and complete or partial 3′ untranslated regions (3′UTRs): pAM 91, complementary to the actin mRNA expressed in adult skeletal muscle (αsk actin); pAF 81, complementary to an actin mRNA that is accumulated in fetal skeletal muscle and is the major transcript in adult cardiac muscle (αc actin); and pAL 41, identified as complementary to a β nonmuscle actin mRNA on the basis of its 3′UTR sequence. As in other species, the protein sequences of these isoforms are highly (>93%) conserved, but the three mRNAs show significant divergence (13.8–16.5%) at silent nucleotide positions in their coding regions. A nucleotide region located toward the 5′ end shows significantly less divergence (5.6–8.7%) among the three mouse actin mRNAs; a second region, near the 3′ end, also shows less divergence (6.9%), in this case between the mouse β and αsk actin mRNAs. We propose that recombinational events between actin sequences may have homogenized these regions. Such events distort the calculated evolutionary distances between sequences within a species. Codon usage in the three actin mRNAs is clearly different, and indicates that there is no strict relation between the tissue type, and hence the tRNA precursor pool, and codon usage in these and other muscle mRNAs examined. Analysis of codon usage in these coding sequences in different vertebrate species indicates two tendencies: increases in bias toward the use of G and C in the third codon position in paralogous comparisons (in the order αc), and in orthologous comparisons (in the order chicken < rodent < man). Comparison of actin-coding sequences between species was carried out using the Perler method of analysis. As one moves backward in time, changes at silent sites first accumulate rapidly, then begin to saturate after −(30–40) million years (MY), and actually decrease between −400 and −500 MY. Replacements or silent substitutions therefore cannot be used as evolutionary clocks for these sequences over long periods. Other phenomena, such as gene conversion or isochore compartmentalization, probably distort the estimated divergence time.

Comparison of three actin-coding sequences in the mouse; Evolutionary relationships between the actin genes of warm-blooded vertebrates

https://www.cell.com/cell/pdf/0092-8674(82)90024-1.pdf

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

Number and organization of actin-related sequences in the mouse genome

We describe the structure and transcriptional activity of the 5' portion of the alpha-cardiac actin gene of BALB/c mice. Southern blotting and DNA sequencing reveal that the promoter and first three exons of the gene are present as perfect repeats in a direct duplication of 9.5 kbp situated immediately upstream of the gene. Both promoters are active in adult cardiac tissue. Transcripts from the partial gene duplication give rise to novel RNAs that are spliced correctly in the actin region and polyadenylated. The level of mature alpha-cardiac actin mRNA is only 16.5% that found in mice that do not possess the duplication. This is due, at least in part, to interference at the transcriptional level. Transcripts from the alpha-skeletal actin gene accumulate to abnormally high levels in the hearts of such mutant mice. This result suggests tight regulatory coupling for this actin gene pair.

A 5' duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue.

Serge Alonso

Papers

Comparison of three actin-coding sequences in the mouse; Evolutionary relationships between the actin genes of warm-blooded vertebrates

A fetal skeletal muscle actin mRNA in the mouse and its identity with cardiac actin mRNA

Number and organization of actin-related sequences in the mouse genome

A 5' duplication of the alpha-cardiac actin gene in BALB/c mice is associated with abnormal levels of alpha-cardiac and alpha-skeletal actin mRNAs in adult cardiac tissue.

Embryonic and fetal myogenic programs act through separate enhancers at the MLC1F/3F locus.