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Characterization of a Novel Human Type II Epithelial Keratin K1b, Specifically Expressed in Eccrine Sweat Glands Lutz Langbein,Ã Michael A. Rogers,w Silke Praetzel,Ã Bernard Cribier,z Bernard Peltre,z Nikolaus Gassler,y and Ju¨ rgen Schweizerw ÃDivision of Cell Biology and wSection of Normal and Neoplastic Epidermal Differentiation, German Cancer Research Center, Heidelberg, Germany; zDepartment of Dermatology, University of Strasbourg, Strasbourg, France; yInstitute of Pathology, University of Heidelberg, Heidelberg, Germany In this study, we show that a novel human type II epithelial keratin, K1b, is exclusively expressed in luminal duct cells of eccrine sweat glands. Taking this luminal K1b expression as a reference, we have used antibodies against a plethora of epithelial keratins to systematically investigate their expression in the secretory globule and the two- layered sweat duct, which was divided into the intraglandular, intradermal, and intraepidermal (acrosyringium) segments, the latter being further subdivided into the sweat duct ridge and upper intraepidermal duct. We show that (i) each of the eccrine sweat gland tissue compartments expresses their own keratin patterns, (ii) the peripheral and luminal duct layers exhibit a sequential keratin expression, with both representing self-renewing cell layers, (iii) the intradermal duct and the sweat duct ridge display hitherto unknown length variations, and (iv) out of all cell layers, the luminal cell layer is the most robust layer and expresses the highest number of keratins, these being concentrated at the apical side of the cells to form the cuticle. We provide evidence that the cellular and inter- cellular properties of the peripheral and the luminal layers reflect adaptations to different functions. Key words: cytoskeleton/differentiation/gene expression/intermediate filaments J Invest Dermatol 125:428 –444, 2005 The genomic characterization of the complex human type I II domain contained only four novel epithelial keratin genes. and type II keratin multigene families on chromosomes Three of these genes had already been identified in 2001, 17q21.2 and 12q13.13, respectively, has involved the work and, at that time, were designated K6l, K1b, and K5b, re- of many laboratories over more than 20 y. Progressing from spectively (Hesse et al, 2001). The fourth gene was discov- the elucidation of single genes or small groups of genes, ered only recently and called Kb20 (Hesse et al, 2004; recently, sequences of larger regions of both domains, Rogers et al, 2004b). which, in addition to epithelial keratin genes, contained the Our laboratories have decided to successively elucidate hair keratin genes expressed in hard keratinizing structures, the expression patterns of the various novel type I and type have also been explored (Rogers et al, 1998, 2000; Lang- II keratin genes. As in previous studies their products had bein et al, 1999, 2001). Considerable progress has been escaped detection by 1- and 2DE gel electrophoresis, it is recently made by the Human Genome Sequencing Con- obvious that, for each of them, either a highly restricted sortium, which has resulted in the completion of a human expression pattern exists, or a particularly weak expression genome reference sequence (The Human Genome Se- in the various epithelial structures of the human body. In this quencing Consortium, 2004). This, coupled with previous study, we undertook the characterization of the type II ker- bioinformatic studies, has led to the elucidation of the entire atin K1b. We show that this keratin is exclusively expressed type I and type II human keratin gene domains (Hesse et al, in the ductal portion of human eccrine sweat glands, and 2001, 2004; Rogers et al, 2004a, b). present a detailed description of the overall keratin expres- The type I keratin gene family contained seven novel sion in this small epidermal adnexal organ. members for which detailed expression studies were still lacking. These comprised five epithelial keratin genes and two hair keratin genes, the latter being separated from the Results previously described cluster of nine functional hair keratin genes (Rogers et al, 1998) by several multigene families Properties and cDNA sequence characteristics of kera- coding for hair keratin-associated proteins (Rogers et al, tin K1b We have previously reported that the K1b keratin 1998, 2001, 2004a; Hesse et al, 2004). In contrast, the type consists of 578 amino acid residues and exhibits a calcu- lated molecular weight of 61.8 kDa (Rogers et al, 2004b). Sequentially, it is highly related to the epidermal differenti- ation-specific keratin K1 (Fig 1A), and hence, evolutionary Abbreviations: IIF, indirect immunofluorescence; ISH, in situ hy- bridization; mab, monoclonal antibody; PBS, phosphate-buffered tree construction, based on the sequences of the a-helical saline rod domains of the known human type II keratins, reveals a Copyright r 2005 by The Society for Investigative Dermatology, Inc. 428 125 : 3 SEPTEMBER 2005 K1b IN SWEAT GLAND DUCTS 429 Figure 1 Comparison of keratin K1b and K1 cDNA sequences and phylogenetic tree. (A) The K1b amino acid sequence is highly homol- ogous with that of K1 in particular in its a- helical rod domains (arrows) but also in its non-a-helical head and tail domains. The underlined sequence motif was used for the generation of an antibody against K1b. As- terisks below a sequence indicate amino acid identity; dots, amino acid homology. (B) Both proteins form a separate branch in the phylogenetic tree constructed on the basis of the rod domain sequences of all known type II keratins. Asterisks in (B) indi- cate the current designation of these kera- tins; p, polymorphic variants (Rogers et al, 2004b). 430 LANGBEIN ET AL THE JOURNAL OF INVESTIGATIVE DERMATOLOGY co-segregation of K1b with K1 (Fig 1B, Rogers et al, 2004b). the K1b cDNA or by indirect immunofluorescence (IIF) mi- Moreover, the K1 and K1b genes are directly neighbored croscopy, using the K1b antiserum prepared against an within the type II keratin gene domain on chromosome aminoterminal peptide of this keratin. These sections in- 12q13.13 (Hesse et al, 2004; Rogers et al, 2004b). Besides cluded glabrous plantar skin whose epidermis, in addition to highly conserved rod domains, the K1b and K1 keratins strong K1 expression, was known to express additional exhibit a highly conserved non-a-helical H1 subdomain and keratins, such as K9 and K2e in differentiating cells (Lang- share distinct glycine-rich sequence stretches in the head bein et al, 1993; Swensson et al, 1998). Although virtually no and tail domains (Fig 1A). A 15 aminoacid oligopeptide, ISH and IIF label was seen in any type of epidermis (cf. Figs derived from the head domain (Fig 1A, underlined), was 3 and 4) nor in any tissue compartment of the complex hair used for the generation of a specific K1b antiserum in follicle (results not shown), rather surprisingly, we observed guinea-pigs. a strong, positive label in a distinct region of eccrine sweat glands. Expression characteristics of K1b Previous northern blot Anatomically, this smallest epidermal appendage con- analyses of a wide range of human tissues using a 30-non- sists of a simple tube, originating blindly in the reticular der- coding probe derived from the K1b gene demonstrated the mis and extending to its opening at the surface of the presence of extremely low amounts of K1b transcripts in epidermis (Fig 2). Eccrine sweat glands can basically be body skin (Rogers et al, 2004b). In contrast, whereas west- divided into two main segments: a proximal secretory glob- ern blots of cytoskeletal extracts of body skin from various ule and a distal excretory duct. The secretory globule is locations clearly revealed reactive bands using K1 or K5 composed of three distinct cell types: alternating inner se- antibodies, no selective K1b protein band could be detect- cretory (clear) and mucoid (dark) cells, occurring in approx- ed when these extracts were incubated with the K1b an- imately equal number and surrounded by spindle shaped, tibody (results not shown). Collectively, these data indicated discontinuous myoepithelial cells that are presumed to that K1b must be a member of the keratin family that is possess contractile properties (Fig 2/1-2). The ductal unit, either extremely weakly or very specifically expressed in which is generally built up of an outer layer of cuboidal pe- restricted areas of the skin. Conceptually, we favored the ripheral cells and an inner layer of luminal cells, can be idea that, given the high sequence homology of K1b with subdivided into four segments: the proximal intraglandular K1, the keratin might be expressed in close association with duct, which is as coiled as the secretory portion, and with K1 in differentiating epidermal cells. We therefore analyzed which it is continuous and invariably mingled in tissue sec- skin sections of various body sites either by in situ hybrid- tions through the secretory globule (Fig 2/1-2). The intra- ization (ISH), using a specific 30-noncoding region probe of glandular duct is followed by the intradermal duct, often Figure 2 Schematic presentation of an eccrine sweat gland and its main tissue segments. Numbers on the left-hand side denote the various segments of a sweat gland in the following order: (1-2), glandular globule, containing secretory (1) and intraglandular duct (2) portions, including the transitional segment between the two segments; (3), intradermal duct; and (4) and (5), intraepidermal duct (acrosyringium) comprising the lower sweat duct ridge (4) and the upper spiralled intraepidermal duct (5). Modified drawing from Abenoza and Ackerman (1990, p 44). 125 : 3 SEPTEMBER 2005 K1b IN SWEAT GLAND DUCTS 431 Figure 3 Demonstration of keratin K1b mRNA and protein by in situ hybridization (ISH) and indirect immunofluorescence (IIF) microscopy. (A–E) ISH reveals specific K1b transcripts (red) in luminal cells (lc) of the intraglandular (igd), intradermal (idd), and intraepidermal/acrosyringial (ied/ac) duct of eccrine sweat glands of plantar skin.

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