Nf1 tumor suppressor in skin:

Expression in response to tissue trauma and in cellular differentiation

Heli Ylä-Outinen

Department of Anatomy and Cell Biology, University of Oulu
Department of Medical Biochemistry, University of Turku2002

Abstract

Type 1 neurofibromatosis (NF1) syndrome is caused by a mutation of the NF1 gene. NF1 protein (neurofibromin) contains a domain which is related to the GTPase activating protein (GAP) and accelerates the switch of active ras-GTP to inactive ras-GDP. The clinical symptoms of NF1 patients include e.g. the formation of benign neurofibroma tumors and hyperpigmented lesions of the skin. The NF1 protein has been referred to as a tumor suppressor since cells of malignant schwannomas of NF1 patients may display loss of heterozygosity of the NF1 gene.

In the present study, the expression of the NF1 gene was investigated during tissue repair in human skin. Elevated NF1 protein levels were seen in a fibroblastic cell population of healing wounds. In vitro studies were designed to investigate NF1 expression in dermal fibroblasts under the influence of growth factors that are operative during wound healing. Platelet-derived growth factor (PDGF) isoforms AB and BB and transforming growth factor β 1 (TGFβ 1) elevated NF1 mRNA levels in cultured dermal fibroblasts. In further studies, histological examination on apparently healthy skin of NF1 patients revealed frequently small masses of neurofibromatous tissue at the vicinity of hair follicles. Thus, action of the NF1 gene appears to be an integral part of normal tissue repair. Enhanced NF1 tumor suppressor expression may serve to limit excessive fibrosis in wound healing.

As ras proteins play a role in the regulation of cell differentiation and formation of cell junctions, the functional expression of NF1 protein was elucidated using differentiating keratinocytes as an in vitro model system. The results demonstrate that an intense NF1 tumor suppressor signal on intermediate filaments was temporally limited to the period in which the formation of desmosomes takes place. In analogy to NF1 protein, a rapid elevation of NF1 mRNA level was detected following initiation of differentiation. Interestingly, NF1 mRNA hybridization signal polarized towards cell-cell contact zones. This finding recognizes a potential way for post-transcriptional modification of NF1 expression and targeting of translation to subplasmalemmal location. The results demonstrate that the function of NF1 is associated with the formation of cell junctions, and thus to cellular communication.

Dedication

Table of Contents

Acknowledgements

Abbreviations

List of original articles

1. Introduction

2. Review of the literature

2.1. NF1 tumor suppressor / histogenesis control factor

2.2. NF1 gene

2.3. NF1 mRNA

2.3.1. NF1 mRNA editing
2.3.2. Unequal expression of NF1 gene alleles
2.3.3. Summary of NF1 RNA processing

2.4. NF1 protein (neurofibromin)

2.4.1. Biology of Ras-proteins

2.5. Type 1 neurofibromatosis syndrome (NF1)

2.5.1. Neurofibromas
2.5.2. Different types of neurofibromatoses

2.6. Skin

2.6.1. Developing skin
2.6.2. Cutaneous wound healing
2.6.3. Cell junctions

2.7. Cellular transport of mRNA

3. Aims of the present study

4. Materials and methods

4.1. Materials (I-IV)
4.2. Methods (I-IV)

5.1. Antiserum NF1as159 against carboxyterminus of NF1 protein (III)

5.2. NF1 tumor suppressor expression in human skin (I, III)

5.2.1. NF1 protein is expressed in normal human skin (I, III)
5.2.2. Altered NF1 protein expression in the healing wounds (I)

5.3. Microscopic changes in the skin of NF1 patients (II)

5.4. NF1 tumor suppressor expression in cultured fibroblasts (I, IV)

5.4.1. NF1 mRNA is expressed in cultured fibroblasts (I, IV)
5.4.2. Up-regulation of NF1 mRNA by selected growth factors (I)

5.5. NF1 tumor suppressor expression in cultured keratinocytes (III, IV)

5.5.1. NF1 protein is expressed in cultured keratinocytes (III, IV)
5.5.2. NF1 protein expression during the cellular differentiation (III)
5.5.3. Targeting of NF1 mRNA in keratinocytes

6. Discussion

6.1. Methodological aspects

6.1.1. Tissues and cell cultures
6.1.2. NF1-specific antibodies

6.2. Discussion of the results

6.2.1. NF1 protein in skin
6.2.2. Elevation of NF1 mRNA expression by growth factors
6.2.3. Role of NF1 protein during cellular differentiation
6.2.4. Expression and translocation of NF1 mRNA during differentiation

7. Conclusions

List of Tables
1. Diagnostic criteria for NF1 (Stumph et al. 1988, Gutmann et al. 1997).
2. Classification of neurofibromatoses (modified from Viskochil & Carey 1994).
3. The tissues and cell lines used are listed below and described in detail in the original publications (I-IV). All tissue samples were collected with appropriate approvals from the ethical committees of the respective universities and university hospitals.
4. The tools for protein analyses.
5. The nucleic acid probes used in Northern hybridizations, RT-PCR, RNase protection assays and ISH.
6. The methods used in the original communications (I-IV).

List of Figures
1. Schematic representation of NF1 gene structure. The transcription start site is marked by an arrow. The transcription stop site and polyadenylation site are marked by an octagon. The GRD is marked, spanning exons 21-27a. The alternatively spliced exons are shown in gray. The three embedded genes within the intron 27b are transcribed to opposite direction (arrow). The asterisk represents the site of mRNA processing. Bar, 1000 bp (introns are not in scale). The figure has been modified from an article by Viskochil (1999b).
2. Alternative splicing of the NF1 RNA. A diversity of NF1 mRNA isoforms are generated by alternative splicing of the exons 9br, 23a, and 48a. The potential RNA editing site is shown with asterisk. The gray numbers indicate the nucleotides in RNA sequence. The N-isoforms 10 and 11 are not demonstrated in the figure. The figure is modified from an article written by Skuse & Cappione (1997).
3. The expression of NF1 gene. The mechanisms affecting NF1 protein levels are illustrated. The top horizontal arrows demonstrate a path of the normal NF1 gene expression. The second path represent the potential effect of the 3’UTR protein-binding regions (PBR) on mRNA stability, translatability, intracellular localization, and the efficiency of translation. The third horizontal route represents the NF1 mRNA editing or biallelic DNA mutation. The bottom path represent the effect of genomic mutations on protein level or on the functional properties, or the unequal allelic expression resulting from a single mutation in the NF1 gene. The figure has been modified from an article by Skuse & Cappione (1997).
4. The functions of NF1 tumor suppressor. NF1 protein acts as a negative regulator of the Ras. The GRD of the NF1 protein accelerates the switch of active Ras-GTP to an inactive Ras-GDP. The NF1 protein has also been shown to interact with cytoskeleton and to be involved in the adenylyl cyclase / PKA pathway. Growth signals (ligand coupled to receptor) activate guanine nucleotide exchange factors (GEFs), which enables GTP binding to Ras-proteins. Signaling pathways downstream of Ras-GTP include phosphatidylinositol-3-kinase (PI3-kinase), Ral-Rac-Rho-, and raf-MEK-ERK kinase cascades.
5. Normal peripheral nerve and a model of tumor development. A) Structure of a normal peripheral nerve. The epineurium, perineurium and constitute the nerve sheath. B) Benign neurofibromas comprise multiple cell types. The structure of the nerve sheath is disrupted and abundant collagenous material is produced. C) LOH of NF1 gene leads to malignancy (neurofibrosarcoma). The figure modified from Cichowsky & Jacks (2001) and based on the findings presented in Peltonen et al. (1984).
6. Epidermal appendages (glands, hair, nails, and mammary glands) develop from epithelium during the embryonic period (Larsen 1993, Holbrook 1997).
7. Schematic representation of cell junctions in simple epithelium.

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