Howard Green - US grants

The protein involucrin is a precursor of the cross-linked envelope that forms late in the terminal differentiation of the human epidermal keratinocyte. This envelope is the most resistant structure in the skin and is the ultimate barrier between the body and the external environment. Involucrin synthesis begins when the keratinocyte reaches a position about halfway through the spinous layer of the epidermis, since that is the point at which the mRNA appears in the cytoplasm. Disorders in terminal differentiation occurring in neoplastic and pre-neoplastic conditions lead to abnormal patterns of involucrin synthesis that can be recognized by immunohistological methods. With a view to understanding how the synthesis of involucrin is regulated, we have isolated cDNA and genomic clones encoding this protein. We have obtained enough sequence data to establish that involucrin is a unique protein whose structure has probably never before been encountered. It possesses a repeating sequence of 10 amino acid residues. This sequence makes the protein a preferential substrate for cross-linking by the keratinocyte transglutaminase that forms the cell envelope. It is obvious from the repeating structure that involucrin evolved through a series of gene duplications that took place either in primates or their near ancestors. We have been able to prepare tryptic fragments of the protein and in one case have been able to map the fragment by comparing its amino acid sequence with the DNA sequence of genomic and cDNA clones. We hope to establish the entire sequence of the cDNA, and then begin to study the regulation of expression of the gene. (M)

The high morbidity and mortality from extensive burns are due mainly to bacterial invasion. This can be prevented by early closure of the burn wounds with autologous skin grafts. In patients with burns of large area, this strategy cannot be fully realized because of the lack of adequate donor sites for grafting. We have demonstrated that it is possible (1) to take a small sample of full thickness epidermis from a badly burned patient and grow the cells in culture using methods we have worked out over the past 10 years, so as to obtain enough cultured epithelium to cover the entire body surface. (2) To prepare the cultured epithelium in a form suitable for grafting. (3) To apply such grafts to humans under clinical conditions, and obtain high frequency of take. (4) To regenerate by this method epidermis covering over 50% of body surface and possessing good quality over moderate to long term. We propose to advance the basic and applied aspects of this system in order to provide living autologous epidermal coverage for any kind of epidermal defect in the human. This procedure is to be evaluated as a definitive solution to the problem of covering in burns of large area. The research will cover short and long term study of the quality of the epidermis regenerated, optimization of the methods of preparing, applying and caring for the grafts and some basic studies relevant to the proliferative potential of the epidermal keratinocyte and its terminal differentiation.

This work is devoted to the study under simple culture conditions of two differentiating mammalian cell types: 1) the human epidermal cell and its close relatives the keratinocytes of other stratified squamous epithelia; this cell type undergoes a program of terminal differentiation to produce a final stage resembling the corneocyte and 2) a preadipose cell line derived from 3T3 and able to differentiate terminally into adipocytes. For both systems we have a number of proteins that are characteristic of the cell in its terminally differentiated state, together with corresponding cDNA and genomic clones. We plan to examine the transition from precursor state, in which the cells are capable of either long term proliferation (human keratinocyte) or indefinite proliferation (3T3 preadipocyte), to a terminally differentiated state in which further division is impossible. We will examine the appearance of specific markers of differentiation in order to understand how the program of differentiation is coordinated. For the epidermal cell we will use cDNA and genomic clones for involucrin and several of the keratins; for adipose differentiation similar clones for glycerophosphate dehydrogenase and two unidentified marker proteins of 13 and 28 kd. This study will include an analysis of the structure of the corresponding genes, the effects of vitamin A on the differentiation of cultured epidermal cells (the regulation of keratin synthesis and of envelope cross-linking), and the effects of growth hormone in promoting the differentiation of preadipose cells. We will attempt to identify the properties of the colony forming epidermal cell, and the alteration of these properties in neoplastic keratinocytes. We will examine the possible role of IGF-1 (the growth hormone induced polypeptide) on clonal expansion of young differentiating adipose cells. These studies are expected to shed light on how differentiation normally leads to the arrest of cell mutiplication and how loss of this coupling leads to neoplasia.

When preadipose 3T3 cells arrest their growth and interact with an external adipogenic factor, they begin to convert to adipose cells. This is a faithful model of the formation of adipose cells in early developement. In the process of adipose conversion there is an extensive revision of the protein composition of the cytoplasm; this is necessary in order to provide the lipogenic and lipolytic enzymes and accesssory proteins for performance of the differentiated functions of the cell type. Using cDNA clones and ultimately genomic clones for some of the proteins participating in the differentiation, we will study the integrated or programmatic aspects of the adipose conversion, in order to learn how the cell coordinates the expression of this group of genes. Studies of transcription and mRNA formation in preadipose 3T3 cells, in converted 3T3 adipose cells and in cells of another 3T3 line unable to undergo conversion in response to adipogenic factor will enable us to define the order in which the genes respond. In particular, we will perform a kinetic analysis of these events during the course of adipose conversion. The relation of the order of expression of these genes to changes in chromatin structure will be explored. The expression of these genes will be studied in other tissues, such as liver and brain, whose program of differentiation overlaps the program in adipose differentiation. In this way we hope to learn how the expression of a given gene can be integrated into different programs in different cell types. These studies permit an approach to the study of obesity using cultured cells. Analysis of the molecular and cellular changes necessary for the formation of adipose cells from their precursors may be expected to shed light on the genesis of hyperplastic obesity.

DESCRIPTION (adapted from applicant's abstract): Huntington disease and eight other central nervous system diseases are each caused by a protein containing an expanded sequence of polyglutamine. Each protein normally contains a polyglutamine sequence of not exceeding 40 residues but mutational expansion of this sequence in any of the proteins produces neuronal damage and a corresponding disease. If the cause can be precisely stated, the pathogenesis cannot. It must be explained why all the diseases are virtually confined to the nervous system and why aggregates or inclusions form in neurons of the affected parts of the brain. While different explanations have been proposed, our previous work supports the following explanation: 1) any protein bearing an expanded sequence of polyglutamine is an exceptionally active substrate of transglutaminase. 2) neurons undergo transient elevation in Ca++ concentration as part of impulse conduction; such a rise would activate the neuronal transglutaminase. 3) the action of transglutaminase couples huntingtin containing expanded polyglutamine to other proteins and produces insoluble aggregates. The constant formation of covalently cross-linked aggregates is lethal to neurons. Further evidence will be sought to support all of these points by studies of 1 ) the purification of inclusion bodies containing expanded polyglutamine from other cellular components and their analysis for the presence of ( abouty-glutamyl) lysine cross-links, 2) the proteins cross-linked in affected brain. especially tubulin, 3) the presence of ( about-glutamyl) lysine in affected parts of the brain and spinal fluid of patients with Huntington Disease. If the role of transglutaminase can be proven conclusively, a prophylaxis and a therapy can be envisioned in the form of transglutaminase inhibitors.

[unreadable] DESCRIPTION (provided by applicant): Human embryonic stem (ES) cells are now available for the study of the generation of somatic cell types. For this purpose, human ES cells have important advantages over the murine ES cells available earlier. We propose to study the generation of keratinocytes of stratified squamous epithelium from the human ES cells (WA01). ES cells are known to have the capacity to generate such epithelium outside the developing embryo, since the epithelium develops when the ES cells are injected into scid mice. But our main intention is to find conditions promoting differentiation of the ES cells in culture and then to isolate strains of keratinocytes resulting from that differentiation, using 3T3 support. We will examine the factors that influence the development of keratinocytes, using as criterion a quantitative measure of the number obtained, and in this way arrive at the conditions producing maximal yield of keratinocytes. We will identify, when possible, the specific squamous epithelium to which isolated keratinocytes belong. We will attempt to find somatic stem cell precursors of the mature keratinocytes using stem cell markers for their identification. Depending on the nature of the keratinocytes or keratinocyte precursors isolated, practical applications can be readily envisioned. [unreadable] [unreadable]