Transforming Growth Factor-β1 (TGF-β1)-stimulated Fibroblast to Myofibroblast Differentiation Is Mediated by Hyaluronan (HA)-facilitated Epidermal Growth Factor Receptor (EGFR) and CD44 Co-localization in Lipid Rafts

Fibroblast to myofibroblast differentiation drives effective wound healing and is largely regulated by the cytokine transforming growth factor-β1 (TGF-β1). Myofibroblasts express α-smooth muscle actin and are present in granulation tissue, where they are responsible for wound contraction. Our previous studies show that fibroblast differentiation in response to TGF-β1 is dependent on and mediated by the linear polysaccharide hyaluronan (HA). Both the HA receptor, CD44, and the epidermal growth factor receptor (EGFR) are involved in this differentiation response. The aim of this study was to understand the mechanisms linking HA-, CD44-, and EGFR-regulated TGF-β1-dependent differentiation. CD44 and EGFR co-localization within membrane-bound lipid rafts was necessary for differentiation, and this triggered downstream mitogen-activated protein kinase (MAPK/ERK) and Ca²⁺/calmodulin kinase II (CaMKII) activation. We also found that ERK phosphorylation was upstream of CaMKII phosphorylation, that ERK activation was necessary for CaMKII signaling, and that both kinases were essential for differentiation. In addition, HA synthase-2 (HAS2) siRNA attenuated both ERK and CaMKII signaling and sequestration of CD44 into lipid rafts, preventing differentiation. In summary, the data suggest that HAS2-dependent production of HA facilitates TGF-β1-dependent fibroblast differentiation through promoting CD44 interaction with EGFR held within membrane-bound lipid rafts. This induces MAPK/ERK, followed by CaMKII activation, leading to differentiation. This pathway is synergistic with the classical TGF-β1-dependent SMAD-signaling pathway and may provide a novel opportunity for intervention in wound healing.     

Earliest Mexican Turkeys (Meleagris gallopavo) in the Maya Region: Implications for Pre-Hispanic Animal Trade and the Timing of Turkey Domestication

Late Preclassic (300 BC-AD 100) turkey remains identified at the archaeological site of El Mirador (Petén, Guatemala) represent the earliest evidence of the Mexican turkey (Meleagris gallopavo) in the ancient Maya world. Archaeological, zooarchaeological, and ancient DNA evidence combine to confirm the identification and context. The natural pre-Hispanic range of the Mexican turkey does not extend south of central Mexico, making the species non-local to the Maya area where another species, the ocellated turkey (Meleagris ocellata), is indigenous. Prior to this discovery, the earliest evidence of M. gallopavo in the Maya area dated to approximately one thousand years later. The El Mirador specimens therefore represent previously unrecorded Preclassic exchange of animals from northern Mesoamerica to the Maya cultural region. As the earliest evidence of M. gallopavo found outside its natural geographic range, the El Mirador turkeys also represent the earliest indirect evidence for Mesoamerican turkey rearing or domestication. The presence of male, female and sub-adult turkeys, and reduced flight morphology further suggests that the El Mirador turkeys were raised in captivity. This supports an argument for the origins of turkey husbandry or at least captive rearing in the Preclassic.     

Site localization of sialyl Lewis(x) antigen on alpha1-acid glycoprotein by high performance liquid chromatography-electrospray mass spectrometry

A simple, fast and sensitive method was developed to verify the presence of the sialyl Lewis(x) antigen on an N-linked glycoprotein. High performance liquid chromatography-electrospray mass spectrometry (HPLC-ESI/MS) was used to identify which of the five N-linked glycosylation sites of human plasma alpha1-acid-glycoprotein (orosomucoid, OMD) contain the sialyl Lewis(x) antigen. OMD was digested with proteolytic enzymes and analyzed by reversed phase chromatography coupled with on-line ESI/MS. A tandem mass spectrometry experiment was designed to detect the presence of the sialyl Lewis(x) antigen based on the observation of an 803 mass to charge ratio ( m/z ) ion produced in the intermediate pressure region of the ESI interface. The ESI/MS signal at m/z 803 is consistent with an oxonium ion for a glycan structure containing NeuAc, Gal, GlcNAc, and Fuc. The identity of the m/z 803 ion was confirmed by ESI/MS/MS analysis of the m/z 803 fragment ion and comparison with a sialyl Lewis(x) standard. The stereochemistry and linkage positions were assigned using previous NMR analysis but could be determined with permethylation analysis if necessary. The analysis of OMD gave a pattern showing signal for the sialyl Lewis(x) antigen coeluting with each of the five N-linked glycopeptides. The ability to monitor sialyl Lewis(x) expression at each of the five sites is of interest in the study of OMD's role in inflammatory diseases.      

The human breast and the ancestral reproductive cycle

This paper, using modern Darwinian theory, proposes an explanation for the increasingly high incidence of breast cancer found among pre-and post-menopausal women living today in westernized countries. A number of factors have been said to be responsible: genetic inheritance (BRCA-1), diet (specifically the increased consumption of dietary fat), exposure to carcinogenic agents, lifetime menstrual activity, and reproductive factors. The primary aim of this paper is to demonstrate the value of a perspective based on Darwinian theory. In this paper, Darwinian theory is used to explore the possibility that the increased incidence of breast cancer is due primarily to the failure to complete in a timely manner the reproductive developmental cycle, beginning at menarche and continuing through a series of pregnancies and lactation. On the basis of comparative data, we assume that most women in ancestral populations began having children before age 20 or so and tended to remain either pregnant or nursing for most of their adult lives. If a woman did not have a child by age 25 or so, she probably would never have one. Therefore, selection would probably not have acted against deleterious traits, such as cancer, that appeared after that age, just as it does not act against such traits in old age. This article is based upon a paper presented at the Sixth Annual Scientific Meeting of the Human Behavior and Evolution Society, June 18th, 1994, University of Michigan, Ann Arbor. Kathryn Coe is a Ph.D. candidate in anthropology at Arizona State University and project director of an NCI grant focusing on cervical and breast cancer in Hispanic women. Field research for her doctoral dissertation focused on the health, fertility, and culture of the Chachi Indians of the coastal rain forest of Ecuador. Lyle Steadman is an assistant professor of anthropology at Arizona State University. He has conducted research for more than two years among the isolated Hewa of Papua New Guinea. His research interests include evolutionary theory and culture, particularly religion and kinship.     

Selection for low or high primary dormancy in Lolium rigidum Gaud seeds results in constitutive differences in stress protein expression and peroxidase activity

Seed dormancy in wild Lolium rigidum Gaud (annual ryegrass) populations is highly variable and not well characterized at the biochemical level. To identify some of the determinants of dormancy level in these seeds, the proteomes of subpopulations selected for low and high levels of primary dormancy were compared by two-dimensional polyacrylamide gel electrophoresis of extracts from mature, dry seeds. High-dormancy seeds showed higher expression of small heat shock proteins, enolase, and glyoxalase I than the low-dormancy seeds. The functional relevance of these differences in protein expression was confirmed by the fact that high-dormancy seeds were more tolerant to high temperatures imposed at imbibition and had consistently higher glyoxalase I activity over 0-42 d dark stratification. Higher expression of a putative glutathione peroxidase in low-dormancy seeds was not accompanied by higher activity, but these seeds had a slightly more oxidized glutathione pool and higher total peroxidase activity. Overall, these biochemical and physiological differences suggest that L. rigidum seeds selected for low dormancy are more prepared for rapid germination via peroxidase-mediated cell wall weakening, whilst seeds selected for high dormancy are constitutively prepared to survive environmental stresses, even in the absence of stress during seed development.     

Hyaluronan Regulates Bone Morphogenetic Protein-7-dependent Prevention and Reversal of Myofibroblast Phenotype

Hyaluronan (HA) promotes transforming growth factor (TGF)-β1-driven myofibroblast phenotype. However, HA can also have disease-limiting activity. Bone morphogenetic protein-7 (BMP7) is an antifibrotic cytokine that antagonizes TGF-β1, and isolated studies have demonstrated that HA can both mediate and modulate BMP7 responses. In this study, we investigated whether BMP7 can modulate HA in a manner that leads to prevention/reversal of TGF-β1-driven myofibroblast differentiation in human lung fibroblasts. Results demonstrated that BMP7 prevented and reversed TGF-β1-driven myofibroblast differentiation through a novel mechanism. BMP7 promoted the dissolution and internalization of cell-surface HA into cytoplasmic endosomes. Endosomal HA co-localized with the HA-degrading enzymes, hyaluronidase-1 and hyaluronidase-2 (Hyal2). Moreover, BMP7 showed differential regulation of CD44 standard and variant isoform expression, when compared with TGF-β1. In particular, BMP7 increased membrane expression of CD44v7/8. Inhibiting CD44v7/8 as well as blocking Hyal2 and the Na⁺/H⁺ exchanger-1 at the cell-surface prevented BMP7-driven HA internalization and BMP7-mediated prevention/reversal of myofibroblast phenotype. In summary, a novel mechanism of TGF-β1 antagonism by BMP7 is shown and identifies alteration in HA as critical in mediating BMP7 responses. In addition, we identify Hyal2 and CD44v7/8 as new potential targets for manipulation in prevention and reversal of fibrotic pathology.