Expression of 36-kDa microfibril-associated glycoprotein (MAGP-36) in human keratinocytes and its localization in skin

doi:10.1016/S0923-1811(01)00148-7

Journal of Dermatological Science

Volume 28, Issue 1, January 2002, Pages 60-67

https://doi.org/10.1016/S0923-1811(01)00148-7 Get rights and content

Abstract

Microfibril-associated glycoprotein-36 (MAGP-36) is a recently isolated elastin-binding protein and considered to be a member of microfibril-associated glycoproteins (MAGPs). We studied the expression of MAGP-36 in cultured normal human keratinocytes and its localization in the skin. MAGP-36 was found to be expressed in cultured human keratinocytes by Western blot and RT-PCR assays. The levels of MAGP-36 (polypeptide and mRNA) and the number of MAGP-36-producing keratinocytes were greatly increased during Ca²⁺-induced differentiation of keratinocytes. Immunohistochemical studies demonstrated that MAGP-36 colocalized with elastic fibers and formed candelabra like-fibers in the superficial dermis of normal skin. In the elderly skin of sun-exposed region, immunoreactivity of MAGP-36 in the superficial dermis disappeared. In the lesional skin of pseudoxanthoma elasticum which is an elastin-related disorder, immunoreactivity of MAGP-36 was found in the accumulation of disintegrated elastic fibers. The results show that MAGP-36 is a component of elastic fibers in the dermis and co-operates with elastin in normal and diseased conditions.

Introduction

Elastic fibers consist of two morphologically different components: amorphous elastin and 10–12 nm microfibrils. Microfibrils are thought to provide scaffolding for the deposition of elastin and to serve to bind tropoelastin in register for the covalent crosslinking [1]. Recent studies have identified several distinct proteins as components of the microfibrils. They include fibrillin-1 (Fib-1) and fibrillin-2 (Fib-2) [2], [3], [4], microfibril-associated glycoprotein-1 (MAGP-1) and -2 (MAGP-2) [5], [6], [7]. MAGPs are components of the microfibrils and appear to be ubiquitously distributed in all microfibril. The amino acid sequence of MAGP-1 deduced from cDNA sequence suggests the presence of two distinct domains. The amino-terminal half of MAGP-1 is highly acidic, enriched in proline and contains a clustering of glutamine residues. The carboxy-terminal half of the molecule contains 13 cystein residues and has an overall net positive charge. The large number of cysteine residues in the region suggests that disulfide bonds are involved in the interaction with elastin molecules and fibrillin-containing microfibril [8], [9], [10].

A unique MAGP with a molecular weight of 36 kDa (MAGP-36) has been recently isolated from porcine aorta [11], [12], [13] and proposed to be included in the member of MAGPs as MAGP-3. This protein is considered to play an important role in the pathogenesis of connective tissue disorders. For example, human homologue of MAGP-36, abnormal aortic aneurysm-associated protein-40 (AAAP-40) or microfibril-associated glycoprotein-4 (MFAP-4) has been found to be immunoreactive with the serum of the patient with abnormal aortic aneurysms (AAAs) [13], [14], or MFAP-4 gene mutation has been reported in the patients with Smith–Magenis syndrome (SMS) [15].

In the papillary layer of the human dermis, elastin and fibrillin-1 form candelabra-like structures and project perpendicularly to the basal lamina of the dermo-epidermal junction [17]. It has been reported that both proteins are expressed by keratinocytes [18], [19]. Recently we have shown that MAGP-1 is produced by human keratinocyte and colocalizes with elastic fibers [20]. In this report, we have studied the expression of MAGP-36 in cultured human keratinocytes. On immunohistchemical examinations, MAGP-36 was found to form candelabra-like structure in the upper dermis as a component of elastic fibers.

Section snippets

Cell culture

Normal human keratinocytes (NHKs) were purchased from Sanko-Junyaku (Tokyo, Japan) and cultured in serum-free, low-calcium (0.1 mM), modified MCDB 153 keratinocyte basal media containing growth factors (designated as KGM), insulin, epidermal growth factor (EGF), hydrocortisone and bovine pituitary extract (BPE). NHKs at 3rd or 4th culture were used in this experiment. To induce terminal differentiation, NHKs were plated at a density 2×10⁴ per cm² in petri dishes and cultured for 4 days in KGM,

Cultured human keratinocytes potentially express MAGP-36

RT-PCR product performed using MAGP-36-specific primers showed a 340 bp DNA fragment in cultured keratinocytes as well as cultured fibroblasts (Fig. 1A). The fragment was subcloned and subjected to sequencing analysis. The nucleotide sequence was found to be identical to that previously reported [14], [15]. Northern blot analysis using RT-PCR product as a probe showed a unique NAGP-36 bp mRNA in the cultured keratinocytes. The amount of MAGP-36 mRNA was found to be increased during Ca²⁺-induced

Discussion

It is noted that cultured human keratinocytes potentially express MAGP-36, in particular as dimeric form with the size of 70 kDa. This is in contrast to cultured fibroblasts in which major form of MAGP-36 is monomeric. Moreover, the level of MAGP-36 in the cell lysate of cultured keratinocytes was greater than that in the cell lysate of cultured fibroblasts. This is possibly because the metabolic rate of MAGP-36 in keratinocytes is different from fibroblasts.

The expression of MAGP-36 in the

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Expression of 36-kDa microfibril-associated glycoprotein (MAGP-36) in human keratinocytes and its localization in skin

Abstract

Introduction

Section snippets

Cell culture

Cultured human keratinocytes potentially express MAGP-36

Discussion

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Vasc. Surg.

J. Invest. Dermatol.

J. Invest. Dermatol.

Anal. Biochem.

Cell

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Extracellular matrix 4: The elastic fiber

FASEB J.

Fibrillin, a new 350-kD glycoprotein, is a component of extracellular microfibrils

J. Cell. Biol.

Partial sequence of a candidate gene for the Marfan syndrome

Nature.

Linkage of Marfan syndrome and a phenotypically related disorder to two different fibrillin genes

Nature