No abstract

In an attempt to identify new members of the human type II hair keratin family by means of 3'- and 5'-RACE methods and cDNA from anagen hair follicles, we detected a sequence that encoded a hitherto unknown type II cytokeratin. The novel cytokeratin comprises 251 amino acids and exhibits the highest sequence homology with K5. Comparative one- and two-dimensional western blots of keratins from anagen hair bulbs, containing or not containing the outer and inner root sheaths (ORS/IRS), and from footsole epidermis with an antibody against the new cytokeratin, revealed its comigration with K6 and its expression in the ORS/IRS complex. We have therefore named the new cytokeratin K6hf, to distinguish it from the various K6 isoforms and to indicate its expression in the hair follicle. Both in situ hybridization with a K6hf-specific cRNA probe and indirect immunofluorescence with the K6hf antibody showed that K6hf is exclusively expressed in the so-called "companion layer" of the hair follicle, a single layered band of flat and vertically oriented cells between the cuboidal ORS cells and the IRS that stretches from the lowermost bulb region to the isthmus of the follicle. Concomitant K17 and K16 expression studies showed that besides suprabasal ORS cells, these cytokeratins are sequentially expressed subsequent to K6hf in companion cells above the hair bulb. Our study confirms the view of a vertically oriented companion layer differentiation. The clearly delayed K17 and K16 expression relative to that of K6hf in companion cells most probably excludes these keratins as possible type I partners of K6hf and suggests the existence of a still unknown type I partner of its own. Thus, not only morphologically but also biochemically, the companion layer is different from the ORS and can therefore be regarded as an independent histologic compartment of the hair follicle.

Low stringency screening of a human P1 artificial chromosome library using a human hair keratin-associated protein (hKAP1.1A) gene probe resulted in the isolation of six P1 artificial chromosome clones. End sequencing and EMBO/GenBank TM data base analysis showed these clones to be contained in four previously sequenced human bacterial artificial chromosome clones present on chromosome 17q12-21 and arrayed into two large contigs of 290 and 225 kilobase pairs (kb) in size. A fifth, partially sequenced human bacterial artificial chromosome clone data base sequence overlapped and closed both of these contigs. One end of this 600-kb cluster harbored six gene loci for previously described human type I hair keratin genes. The other end of this cluster contained the human type I cytokeratin K20 and K12 gene loci. The center of the cluster, starting 35 kb downstream of the hHa3-I hair keratin gene, contained 37 genes for high/ultrahigh sulfur hair keratinassociated proteins (KAPs), which could be divided into a total of 7 KAP multigene families based on amino acid homology comparisons with previously identified sheep, mouse, and rabbit KAPs. To date, 26 human KAP cDNA clones have been isolated through screening of an arrayed human scalp cDNA library by means of specific 3-noncoding region polymerase chain reaction probes derived from the identified KAP gene sequences. This screening also yielded four additional cDNA sequences whose genes were not present on this gene cluster but belonged to specific KAP gene families present on this contig. Hair follicle in situ hybridization data for single members of five different KAP multigene families all showed localization of the respective mRNAs to the upper cortex of the hair shaft.

In this study we have characterized a novel human type II keratin, hK6irs1, which is specifically expressed in the inner root sheath of the hair follicle. This keratin represents the ortholog of the recently described mouse inner root sheath keratin mK6irs. The two keratins were highly related and migrated at the same height as keratin 6 in two-dimensional gel electrophoresis. Both RNA in situ hybridization and indirect immunofluorescence studies of human hair follicles demonstrated hK6irs1 expression in the Henle and Huxley layers as well as in the cuticle of the inner root sheath. In all three layers, the expression of hK6irs1 mRNA and protein began simultaneously in adjacent cells of the lowermost bulb above the germinative cell pool. Higher up in the follicle, the detection limits for both hK6irs1 mRNA and protein precisely coincided with the asynchronous onset of abrupt terminal differentiation of the Henle layer, inner root sheath cuticle, and Huxley layer. Mainly above the level of terminal Henle cell differentiation, both indirect immunofluorescence and immunoelectron microscopy revealed the occurrence of distinct Huxley cells that developed pseudopodal hK6irs1-positive extensions passing through the fully keratinized Henle layer. These outwardly protruding foot processes abutted upon cells of the companion layer, with which they were connected by numerous desmosomes. These specialized Huxley cells have previously been termed "Flügelzellen", which means "winged cells", with reference to their characteristic foot processes. We provide evidence that, together with Henle cells, Flügelzellen ensure the maintenance of a continuous desmosomal anchorage of the companion layer along the entire inner root sheath. This tightly connected companion layer/inner root sheath unit provides an optimal molding and guidance of the growing hair shaft.