Stephanie R. Edmondson

Interferon-resistant Human Melanoma Cells Are Deficient in ISGF3 Components, STAT1, STAT2, and p48-ISGF3γ

The mechanism of IFN resistance was examined in three long-term cell lines, SK-MEL-28, SK-MEL-3, and MM96, exhibiting significant variation in responsiveness to the antiproliferative and antiviral effects of type I IFNs. The JAK-STAT components involved in IFN signal transduction were analyzed in detail. After exposure to IFN, activation of the IFN type I receptor-linked tyrosine kinases, JAK-1 and TYK-2, was detected at similar levels in both IFN-sensitive and IFN-resistant cell types, indicating that IFN resistance did not result from a deficiency in signaling at the level of receptor-associated kinase activation. However, analysis of ISGF3 transcription factor components, STAT1, STAT2, and p48-ISGF3γ, revealed that their expression and activation correlated with cellular IFN responsiveness. The analysis was extended to also include IFN-sensitive primary melanocytes, three additional IFN-resistant melanoma cell lines, and seven cell cultures recently established from melanoma patient biopsies. It was consistently observed that the most marked difference in ISGF3 was a lack of STAT1 in the resistant versus the sensitive cells. Transfection of the IFN-resistant MM96 cell line to express increased levels of STAT1 protein partially restored IFN responsiveness in an antiviral assay. We conclude that a defect in the level of STAT1 and possibly all three ISGF3 components in IFN-resistant human melanoma cells may be a general phenomenon responsible for reduced cellular responsiveness of melanomas to IFNs.

Reversal of epidermal hyperproliferation in psoriasis by insulin-like growth factor I receptor antisense oligonucleotides.

Epidermal hyperplasia is a key feature of the common skin disorder psoriasis. Stimulation of epidermal keratinocytes by insulin-like growth factor I (IGF-I) is essential for cell division, and increased sensitivity to IGF-I may occur in psoriasis. We hypothesized that inhibition of IGF-I receptor expression in the psoriasis lesion would reverse psoriatic epidermal hyperplasia by slowing the rate of keratinocyte cell division. Here we report the use of C5-propynyl-dU,dC-phosphorothioate antisense oligonucleotides to inhibit IGF-I receptor expression in keratinocytes. We identified several inhibitory antisense oligonucleotides and demonstrated IGF-I receptor inhibition in vitro through an mRNA targeting mechanism. Repeated injection of these oligonucleotides into human psoriasis lesions, grafted onto nude mice, caused a dramatic normalization of the hyperplastic epidermis. The findings indicate that IGF-I receptor stimulation is a rate-limiting step in psoriatic epidermal hyperplasia and that IGF-I receptor targeting by cutaneous administration of antisense oligonucleotides forms the basis of a potential new psoriasis therapy.

Expression of insulin-like growth factor binding protein-3 (IGFBP-3) in human keratinocytes is regulated by EGF and TGF?1

Insulin‐like growth factor‐I (IGF‐I) is essential for normal epidermal homeostasis; however, the role of IGF binding proteins (IGFBPs), regulators of IGF action, remains unclear. Here we examine the regulation of human keratinocyte‐produced IGFBPs by epidermal growth factor (EGF), transforming growth factor beta 1 (TGFβ1), and IGF‐I, growth factors known to be active in skin. In the absence of added growth factors, IGFBP‐3 was the major binding protein secreted into the medium by primary keratinocytes. Addition of EGF or TGFβ1 to keratinocyte cultures resulted in a significant decrease in IGFBP‐3 abundance in conditioned medium when compared with control, untreated cells. Specifically, EGF (50 ng/ml) and TGFβ1 (50 ng/ml) reduced IGFBP‐3 abundance to 15 ± 6% and 22 ± 9%, respectively. Using Northern blot analysis, we found EGF and TGFβ1 (50 ng/ml) to reduce IGFBP‐3 mRNA levels in keratinocytes to 51 ± 12% and 50 ± 38%, respectively, when compared with control, untreated cells. Treatment with IGF‐I or its analogue des(1‐3)IGF‐I did not lead to any consistent change in IGFBP‐3 abundance. However, both IGF‐I and des(1‐3)IGF‐I at 100 ng/ml led to a modest increase in IGFBP‐3 mRNA levels in keratinocytes, suggesting posttranscriptional regulation of IGFBP‐3 abundance. We propose that local modulation of IGFBP‐3 abundance may represent another level of regulation of growth factor action in the epidermis, where EGF and TGFβ1 and possibly other local growth factors specifically regulate the availability of IGF‐I to its keratinocyte receptors. J. Cell. Physiol. 179:201–207, 1999.

Localization of the Insulin-like Growth Factor System in a Rat Model of Heart Failure Induced by Myocardial Infarction

Although cardiac effects of growth hormone (GH) and insulin-like growth factor (IGF)-I have been reported in experimental models of heart failure and in human dilated cardiomyopathy, the IGF system has not been comprehensively assessed in the failing heart. We therefore localized the IGF system in the left ventricle during congestive heart failure after myocardial infarction (Ml) in the rat. The left anterior descending coronary artery was ligated in adult female Sprague-Dawley rats and hearts were examined after 6 months when congestive heart failure had developed. In situ hybridization histochemistry was used to localize mRNA for the components of the IGF system in the left ventricle of sham and congestive heart failure animals. We were able to detect changes in the spatial distribution of mRNA for IGF-I and IGF binding proteins 3, 4, 5, and 6 in the left ventricle during congestive heart failure after Ml. IGF-I and the binding proteins were predominantly increased in the infarct/peri-infarct area of the left ventricle. Other components of the IGF system were indistinguishable from the low to undetectable levels in sham-operated rats. These results demonstrate that the IGF system is altered in the failing heart and suggest that the IGF system plays an important role in the response of the heart to Ml and consequent failure.

UV induced responses of the human epidermal IGF system: impaired anti-apoptotic effects of IGF-I in HaCaT keratinocytes.

The insulin-like growth factor receptor (IGF-IR) is critical in epidermal development and IGF binding protein-3 (IGFBP-3), a modulator of cellular activity with or without IGF-dependence, co-localises with epidermal IGF-IRs. We have investigated whether the greater UV susceptibility of a human keratinocyte cell line (HaCaT) in comparison to normal human keratinocytes (NHKs) may involve differences in the IGF system. At 24 h after UV (960 mJ/cm2 UVB), in comparison to NHKs, HaCaT keratinocytes exhibited significantly higher levels of apoptosis, refractoriness to IGF-I treatment and reduced IGF-IR phosphorylation. Secreted, intact IGFBP-3 (38–42 kDa) and IGFBP-3 mRNA abundance were reduced in HaCaT keratinocytes, but not consistently altered in NHKs. Immunoreactive IGFBP-3 fragments (16–11 kDa) were detected in both UV-exposed cultures. These data suggest that an altered IGF system contributes to HaCaT keratinocyte UV susceptibility and that following UV insult the IGF system may enhance keratinocyte viability and contribute to a return to epidermal homeostasis.