Biomedical subjects

Latha Satish

Publications and source records attributed to Latha Satish.

6 recordsLinked to original sources

PubMed2005

Interferon-inducible protein 9 (CXCL11)-induced cell motility in keratinocytes requires calcium flux-dependent activation of mu-calpain.

Keratinocyte migration is critical to reepithelialization during wound repair. The motility response is promoted by growth factors, cytokines, and cytokines produced in the wound bed, including those that activate the epidermal growth factor (EGF) receptor. The Alu-Leu-Arg-negative CXC chemokine interferon-inducible protein 9 (IP-9; also known as CXCL11, I-TAC, beta-R1, and H-174) is produced by keratinocytes in response to injury. As keratinocytes also express the receptor, CXCR3, this prompted us to examine the role and molecular mechanism by which IP-9 regulates keratinocyte motility. Unexpectedly, as CXCR3 liganding blocks growth factor-induced motility in fibroblasts, IP-9 alone promoted motility in undifferentiated keratinocytes (37 +/- 6% of the level of the highly motogenic EGF) as determined in a two-dimensional in vitro wound healing assay. IP-9 even enhanced EGF-induced motility in undifferentiated keratinocytes (116 +/- 5%; P < 0.05 compared to EGF alone), suggesting two separate mechanisms of action. IP-9-increased motility and -decreased adhesiveness required the intracellular protease calpain. The increases in both motility and calpain activity by IP-9 were blocked by pharmacological and molecular inhibition of phospholipase C-beta3 and chelation of calcium, which prevented an intracellular calcium flux. Molecular downregulation or RNA interference-mediated depletion of mu-calpain (calpain 1) but not M-calpain (calpain 2) blocked IP-9-induced calpain activation and motility. In accord with elimination of IP-9-induced de-adhesion, RNA interference-mediated depletion of calpain 1 but not calpain 2 prevented cleavage of the focal adhesion component focal adhesion kinase and disassembly of vinculin aggregates. In comparison, EGF-induced motility of the same undifferentiated keratinocytes requires the previously described extracellular signal-regulated kinase to the M-calpain pathway. These data demonstrate that while both EGF- and IP-9-induced motility in keratinocytes requires calpain activity, the isoform of calpain triggered depends on the nature of the receptor for the particular ligand. Interestingly, physiological nonapoptotic calcium fluxes were capable of activating mu-calpain, implying that the calcium requirement of mu-calpain for activation is attained during cell signaling. This is also the first demonstration of differential activation of the two ubiquitous calpain isoforms in the same cell by different signals.

Calcium↗

PubMed2003

Aging-related attenuation of EGF receptor signaling is mediated in part by increased protein tyrosine phosphatase activity.

As fibroblasts near senescence, their responsiveness to external signals diminishes. This well-documented phenomenon likely underlies physiological deterioration and limited tissue regeneration in aging individuals. Understanding the underlying molecular mechanisms would provide opportunities to ameliorate these situations. A key stimulus for human dermal fibroblasts are ligands for the epidermal growth factor receptor (EGFR). We have shown earlier that EGFR expression decreases by about half in near senescent fibroblasts (Shiraha et al., 2000, J. Biol. Chem. 275 (25), 19343-19351). However, as the cell responses are nearly absent near senescence, other aging-related signal attenuation changes must also occur. Herein, we show that EGFR signaling as determined by receptor autophosphorylation is diminished over 80%, with a corresponding decrease in the phosphorylation of the immediate postreceptor adaptor Shc. Interestingly, we found that this was due at least in part to increased dephosphorylation of EGFR. The global cell phosphotyrosine phosphatase activity increased some threefold in near senescent cells. An initial survey of EGFR-associated protein tyrosine phosphatases (PTPases) showed that SHP-1 (PTPIC, HCP, SHPTP-1) and PTPIB levels are increased in parallel in these cells. Concomitantly, we also discovered an increase in expression of receptor protein tyrosine phosphatase alpha (RPTPalpha). Last, inhibition of protein tyrosine phosphatases by sodium orthovanadate in near senescent cells resulted in increased EGFR phosphorylation. These data support a model in which, near senescence, dermal fibroblasts become resistant to EGFR-mediated stimuli by a combination of receptor downregulation and increased signal attenuation.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Glu-Leu-Arg-negative CXC chemokine interferon gamma inducible protein-9 as a mediator of epidermal-dermal communication during wound repair.

Normal wound healing is a complex, highly regulated dynamic process that requires co-ordinate responses of both epidermal and dermal compartments. To accomplish the healing process several growth factors, chemokines, and matrix elements signal both cell proliferation and migration during the inflammatory and reparative phases and limit these responses during the remodeling phase. We have found that the Glu-Leu-Arg-negative CXC chemokines interferon gamma inducible protein 10, monokine induced by interferon gamma, and platelet factor 4, limit fibroblast responsiveness to growth factors, but the functioning of these factors in wound healing remains uncertain. We hypothesized that the keratinocyte-derived member of this Glu-Leu-Arg-negative CXC family, interferon gamma inducible protein 9 (IP-9) CXCL11 (also known as I-TAC, beta-R1, and H-174) signals to the dermal compartment to synchronize the re-epithelialization process. Interferon gamma inducible protein 9 was produced after mechanical wounding of a keratinocyte monolayer, suggesting for the first time that this could be a wound response factor. Interferon gamma inducible protein 9 limited epidermal growth factor (EGF)-induced fibroblast motility (57+/-7%) by the same protein kinase A (KA)-mediated inhibition of calpain activation and cell de-adhesion as described for interferon gamma inducible protein 10. Surprisingly, interferon gamma inducible protein 9 enhanced growth factor-induced motility in undifferentiated keratinocytes (137+/-19%) as determined in a two-dimensional in vitro wound healing assay, and interferon gamma inducible protein 9 alone promoted motility in undifferentiated keratinocytes (49+/-10% of epidermal growth factor-induced motility). A stimulated keratinocyte/target cell coculture system revealed that interferon gamma inducible protein 9 acts as a soluble keratinocyte-derived paracrine factor for both fibroblasts and keratinocytes. Further, we found that in both fibroblasts and undifferentiated keratinocytes, interferon gamma inducible protein 9 exerted its action through modulation of a cytosolic protease, calpain. Interestingly, interferon gamma inducible protein 9 increased calpain activity in undifferentiated keratinocytes, whereas the same chemokine inhibited the calpain activity in fibroblasts. This provides for a model whereby redifferentiated basal keratinocytes could limit fibroblast repopulation of the dermis underlying healed wounds while simultaneously promoting re-epithelialization of the remaining provisional wound.

Calpain↗

PubMedDate not supplied

TGF beta-like regulation of matrix metalloproteinases by anti-transforming growth factor-beta, and anti-transforming growth factor-beta 1 antibodies in dermal fibroblasts: Implications for wound healing.

Transforming growth factor beta (TGF-beta) stimulates collagen and matrix metalloproteinase-2 expression and inhibits MMP-1 expression in dermal fibroblasts. Anti-TGF-beta antibodies have been proposed in the prevention of wound scars. The goal of this research was to investigate the regulation of matrix metalloproteinases-1 and -2 expression at the protein, mRNA, and transcriptional levels using an anti-TGF-beta antibody to TGF-beta 1, 2, 3, and 5 (all isoforms), and specifically by an anti-TGF-beta 1 antibody. Both antibodies, though at doses lower than the recommended neutralization dose, stimulated the expression of TGF-beta, and exhibited TGF-beta-like regulation of the matrix metalloproteinases. The antibodies inhibited matrix metalloproteinase-1 protein, mRNA, and promoter activity. The protein levels of matrix metalloproteinase-2 were up-regulated to a greater extent than the matrix metalloproteinase-2 mRNA level by both antibodies. These effects of anti-TGF-beta and anti-TGF-beta 1 antibodies on matrix metalloproteinase regulation were mimicked by exogenous TGF-beta 1 but not rabbit or chicken IgG. We infer that the anti-TGF-beta1 isoform that forms part of the composition of the anti-TGF-beta antibody to all isoforms may be responsible for the feedback stimulation of TGF-beta and the resultant alterations in the expression of the matrix metalloproteinases by the anti-TGF-beta antibodies.

Antibodies↗

PubMedDate not supplied

Gene expression patterns in isolated keloid fibroblasts.

Keloid scars after skin trauma are a significant clinical problem, especially in black populations, in which the incidence of keloids has been estimated at 4-16%. Keloids are abnormal dermal proliferative scars secondary to dysregulated wound healing. Despite several biochemical studies on the role of extracellular matrix proteins and growth factors during keloid formation, we still do not know what molecules and signals induce this change. Fibroblasts are thought to be the major inductive cell for keloid scar formation. The aim of this study was to identify gene expression patterns that characterize keloid fibroblasts; identifying such genetic disequilibrium may shed light on the molecular signaling events responsible for keloid formation. In this study, we performed gene expression analysis of fibroblasts isolated from keloid lesions from three individuals in comparison with the fibroblasts isolated from normal skin using the Affymetrix U133a chip (22,284 genes and expression sequence tags). We found through J5 test score expression analysis that among 22,284 genes, there were 43 genes that were overexpressed and five genes were underexpressed in keloid fibroblasts when compared with dermal fibroblasts from persons without keloids. The overexpression of three genes not previously reported as being up-regulated in keloids (annexin A2, Transgelin, and RPS18) was confirmed by real-time polymerase chain reaction. Certain overexpressed genes were similar to previous biochemical observations on the protein levels of these overexpressed genes during keloid formation. We also report for the first time that a few tumor-related genes are overexpressed in keloid fibroblasts.

Adult↗

PubMedDate not supplied

Identification of differentially expressed genes in scarless wound healing utilizing polymerase chain reaction-suppression subtractive hybridization.

Wound healing in fetal skin is well known to proceed without scarring, whereas adult (postnatal) skin wound healing is accompanied by scar formation. To identify differentially expressed genes during fetal wound (FW) healing, we have used polymerase chain reaction-suppression subtractive hybridization. This technique allows for a comparative analysis across the entire transcriptome of FW vs. unwounded fetal control tissue, including even potentially novel sequences. Our subtractive hybridization protocol identified 15 clones that are overexpressed in healing FWs, and 20 clones that are underexpressed. These include genes with both known and unknown functions. We have confirmed the differential pattern of expression for four of these candidate genes: elongation factor 1 alpha, elongation initiation factor 4e, and two transcripts thus far known only as an expressed sequence tags. With this approach, we have also identified novel genes potentially involved in scarless wound healing.

Animals↗