The catalog of human hair keratins. II. Expression of the six type II members in the hair follicle and the combined catalog of human type I and II keratins.

The human type II hair keratin subfamily consists of six individual members and can be divided into two groups. The group A members hHb1, hHb3, and hHb6 are structurally related, whereas group C members hHb2, hHb4, and hHb5 are rather distinct. Specific antisera against the individual hair keratins were used to establish the two-dimensional catalog of human type II hair keratins. In this catalog, hHb5 showed up as a series of isoelectric variants, well separated from a lower, more acidic, and complex protein streak containing isoelectric variants of hair keratins hHb1, hHb2, hHb3, and hHb6. Both in situ hybridization and immunohistochemistry on anagen hair follicles showed that hHb5 and hHb2 defined early stages of hair differentiation in the matrix (hHb5) and cuticle (hHb5 and hHb2), respectively. Although cuticular differentiation proceeded without the expression of further type II hair keratins, cortex cells simultaneously expressed hHb1, hHb3, and hHb6 at an advanced stage of differentiation. In contrast, hHb4, which is undetectable in hair follicle extracts and sections, could be identified as the largest and most alkaline member of this subfamily in cytoskeletal extracts of dorsal tongue. This hair keratin was localized in the posterior compartment of the tongue filiform papillae. Comparative analysis of type II with the previously published type I hair keratin expression profiles suggested specific, but more likely, random keratin-pairing principles during trichocyte differentiation. Finally, by combining the previously published type I hair keratin catalog with the type II hair keratin catalog and integrating both into the existing catalog of human epithelial keratins, we present a two-dimensional compilation of the presently known human keratins.

Characterization of a cluster of human high/ultrahigh sulfur keratin-associated protein genes embedded in the type I keratin gene domain on chromosome 17q12-21.

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 keratin-associated 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.

Human type I hair keratin pseudogene phihHaA has functional orthologs in the chimpanzee and gorilla: evidence for recent inactivation of the human gene after the Pan-Homo divergence.

In addition to nine functional genes, the human type I hair keratin gene cluster contains a pseudogene, phihHaA (KRTHAP1), which is thought to have been inactivated by a single base-pair substitution that introduced a premature TGA termination codon into exon 4. Large-scale genotyping of human, chimpanzee, and gorilla DNAs revealed the homozygous presence of the phihHaA nonsense mutation in humans of different ethnic backgrounds, but its absence in the functional orthologous chimpanzee (cHaA) and gorilla (gHaA) genes. Expression analyses of the encoded cHaA and gHaA hair keratins served to highlight dramatic differences between the hair keratin phenotypes of contemporary humans and the great apes. The relative numbers of synonymous and non-synonymous substitutions in the phihHaA and cHaA genes, as inferred by using the gHaA gene as an outgroup, suggest that the human hHaA gene was inactivated only recently, viz., less than 240,000 years ago. This implies that the hair keratin phenotype of hominids prior to this date, and after the Pan-Homo divergence some 5.5 million years ago, could have been identical to that of the great apes. In addition, the homozygous presence of the phihHaA exon 4 nonsense mutation in some of the earliest branching lineages among extant human populations lends strong support to the "single African origin" hypothesis of modern humans.

The catalog of human hair keratins. I. Expression of the nine type I members in the hair follicle.

The human type I hair keratin subfamily comprises nine individual members, which can be subdivided into three groups. Group A (hHa1, hHa3-I, hHa3-II, hHa4) and B (hHa7, hHa8) each contains structurally related hair keratins, whereas group C members hHa2, hHa5, and hHa6 represent structurally rather unrelated hair keratins. Antibodies produced against these individual hair keratins, first analyzed for specificity by one- dimensional Western blots of total hair keratins, were used to establish the two-dimensional catalog of the human type I hair keratin subfamily. The catalog comprises two different series of type I hair keratins: a strongly expressed, Coomassie-stainable series containing hair keratins hHa1, hHa3-I/II, hHa4, and hHa5, and a weakly expressed, immunodetectable series harboring hHa2, hHa6 hHa7, and hHa8. In situ hybridization and immunohistochemical expression studies on scalp follicles show that two hair keratins, hHa2 and hHa5, define the early stage of hair differentiation, i.e. hHa5 expression in hair matrix and hHa5/hHa2 coexpression in the early hair cuticle cells. Whereas cuticular differentiation proceeds without the expression of further type I hair keratins, matrix cells embark on the cortical pathway by sequentially expressing hHa1, hHa3-I/II, and hHa4, which are supplemented by hHa6 at an advanced stage of cortical differentiation, and hHa8, which is expressed heterogeneously in cortex cells. Thus, six type I hair keratins are involved in the terminal differentiation of anagen hairs. The expression of hHa7 is conspicuously different from that of the other hair keratins in that it does not occur in the large anagen follicles of terminal scalp hairs but only in central cortex cells of the rare and small follicle type that gives rise to vellus hairs.

A novel human type II cytokeratin, K6hf, specifically expressed in the companion layer of the hair follicle.

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.

Sequences and differential expression of three novel human type-II hair keratins.

As part of a program designed to characterize human hair keratin genes and their expression, we present the cDNA sequences and deduced amino acid sequences of three type-II hair keratins hHb3, hHb5, and hHb6, which by virtue of their amino acid homologies are the orthologs of the previously described sheep wool keratins, K2.10, K2.12, and K.211 [29]. Amino acid sequences comparisons of these keratins, including the previously characterized human K2.9 ortholog hHb1, show extreme conservation not only in the alpha-helices but also in the aminoterminal and proximal carboxyterminal domains. They also demonstrate higher sequence relationships between hHb1, hHb3, and hHb6 as compared to hHb5, which exhibits chain-specific sequences in both the head and tail domains. In situ hybridization studies using specific 3'-probes for the four type-II hair keratins reveal sequential patterns of gene expression in human anagen follicles. Remarkably the onset of hHb5 mRNA synthesis occurs immediately above a small population of matrix cells at the base of the hair bulb and the trichocytes lining the dermal papilla. hHb5 mRNA synthesis extends upward through the matrix and ends in the lower part of the cortex of the hair shaft. In contrast, both hHb1 and hHb3 mRNA synthesis begins simultaneously in the cortex 10-15 cell layers above the apex of the dermal papilla, thus partially overlapping that of hHb5 but continuing to a point well beyond hHb5 in the upper cortex. Synthesis of hHb6 mRNA starts slightly higher than either hHb1 or hHb3 mRNA and proceeds much farther up into the keratogenous zone of the hair shaft. Our study demonstrates that the differentiation of human hair in terms of hair keratin expression begins much earlier than previously assumed, i.e. in lower matrix cells of the hair bulb. This early phase of hair differentiation is followed by a late cortical phase of terminal differentiation which comprises at least three type-II hair keratins in the zone of elongation and the keratogenous zone of the hair shaft.