Effects of vitamin-A status on hamster tracheal epithelium in viva in vitro

Luigi M. De Luca and Elizabeth M. McDowell

This paper highlights what is known about how vitamin A and the retinoids control epithelial morphology and function. The system of choice is the tracheal epithelium of the Syrian golden hamster. This species was originally selected by Saffiotti et al. [1] in their studies of chemical carcinogenesis of the respiratory tract because it is relatively resistant to respiratory infection. The system was then rendered more amenable to in vitro investigation by Sporn et al. [2], who defined conditions for maintaining the trachea in organ explant culture, as well as the tissue's requirement for retinoic acid (RA) in the maintenance of normal mucociliary differentiation. Following in viva observations [3; 4], McDowell et al. have recently used epithelial cell culture techniques to define the mucous cell as the target of vitamin A [5].

In vivo effect of vitamin-A status on the hamster tracheal epithelium

The final effect of nutritional deficiency of vitamin A on the tracheal epithelium is the replacement of ciliated cells and normal mucous cells (fig. 1) by squamoid cells (altered mucous cells), which normally characterize the epidermoid type of differentiation. Figure 2 shows the extent of such replacement, which results in the near occlusion of the tracheal lumen. Accumulation of bacteria and other external material influences survival of the animal, which eventually succumbs to infection.

The terminal stage is preceded by primary effects of the deficient diet. Careful monitoring of the changes in body weight and serum retinal levels allowed the definition of a stage of "minimal morphological change," which preceded the loss of body weight [3; 4; 6]. Measurement of the cell-division rates in the mucous and basal cells during the development of vitamin-A deficiency revealed that the rates (mitotic rates) were lowered in a non-uniform manner. The replication of mucous cells was profoundly reduced compared with that of the basal cells during the stage of "minimal morphological change." This is clearly shown in table 1.

This work leads to the conclusion that vitamin-A deficiency depresses epithelial cell division before epidermoid metaplasia formation is evident. If vitamin-A deficiency continues, however, the altered mucous cells regain the capacity to divide and the epithelium is replaced by flat "squamoid cells." These cells generally arise as a consequence of cell injury, whether caused by carcinogen exposure, mechanical injury, or, as in this case, nutritional deficiency (fig. 3). It is clear, then, that squamoid cells (but not columnar mucous cells) can survive and multiply in the absence of vitamin A. Reversal of squamous metaplasia to the normal mucociliary phenotype of the tracheal epithelium is only possible in the presence of vitamin A or one of its biologically active analogues.

TABLE 1. Proportions and mitotic rates of tracheal basal, mucous, and ciliated cells of control and vitamin-A-deprived hamsters

Cell type	Control	Vitamin-A-deprived	P value
Proportion of total (%)
Basal	28.7 ± 2.5	39.7 ± 2.7	<.0001
Mucous	59.3 ± 2.6	53.1 ± 1.9	<.001
Ciliated	11.0 ± 3.1	6.8 ± 1.8	<.05
Mitotic rate (% of total)a
Basal	0 61 ± 0.13	0.29 ± 0.35	<.062
Mucous	2.42 ± 1.19	0.15 ± 0.10	<.01
Ciliatedb	0	0
Mitotic rate (% of own cell type)c
Basal	2.14 ± 0.48	0.72 ± 0.91	<.01
Mucous	4.06 ± 1.97	0.29 ± 0.2	<.01
Ciliatedb	0	0

Source: Ref. 4 Reproduced by permission.

The data for vitamin-A-deprived cells were derived from epithelia showing minimal changes after five weeks on diet. Foci of stratification and/or epidermoid metaplasia (about 5% of all epithelial cells) were excluded from the analysis.
a. Mean percentage of total number of epithelial cells counted in cross-section of two tracheal rings per hamster (about 1,600 cells). Each hamster received 3H-thymidine and colchicine six hours before sacrifice.
b. Ciliated cells do not divide.
c. Mean percentage of total number of basal cells or mucous cells.

Administration of retinyl acetate to vitamin-A-deficient hamsters, again, primarily affects cell division of the mucous cells (fig. 4). Within three days their cell-division rate returned to normal levels. The number of preciliated cells, which are progeny of replicating mucous cells, was restored to normal levels, but vitamin-A repletion had no effect on the replication of the basal cells.

The first unequivocal conclusion from this in viva work is that vitamin A is necessary to maintain normal rates of mucous cell division in the tracheal epithelium.

FIG. 4. Relationship between the proportion of. preciliated cells and the mitotic rates (M.R.) of mucous cells and basal cells All data represent mean percentages of the total number of epithelial cells counted. Control values were 0.39% for the proportion of preciliated cells and 0.61% and 2.2% for the mitotic rate of basal cells and mucous cells respectively.

In vitro cell culture work

Recent work is] has permitted observation of the effects of vitamin-A depletion in cultured epithelial cells from hamster tracheas. The cells recapitulated the development of normal epithelium during seven days of culture. In the presence of retinoic acid, the mucous cells divided at a high rate and the progeny rapidly matured to fully differentiated mucous and ciliated cells (fig. 5). Smaller cells were also visible in the culture and probably represented basal cells found in viva. The study compared the morphology and cell-division rates of the cells in the presence and absence of retinoic acid. It is clear from figure 6 that deficiency of vitamin A markedly lowered the ability of the larger mucous cells to divide.

FIG. 6. The effect retinoic-acid (RA) depletion on ³H-thymidine-labelled nuclei of small (basal) and large (secretory) tracheal epithelial cells - percentages of each kind of cell. The large cells were very flat in RA- cultures on days 2 and 4, and so it was possible to discriminate between the nuclei of the two kind of cells

When cells grew in the absence of retinoic acid on collagen substrate, they failed to mature into normal columnar mucous cells and, instead, showed the squamous type of differentiation characteristic of vitamin-A deficiency in vivo and of organ-cultured epithelium (fig7).

This in vitro work confirms the notation that retinoic acid in required for cell division and differentation of mucous cells.

The control of mucus production by vitamin A

In conjunction with the lowered rate of mucous cell division, vitamin-A deficiency also causes a decrease in periodic-acid Schiff base (PAS)-positive cells, which indicates less production of mucus. Whether this precedes or follows the decrease in cell division is unclear, but it certainly appears to precede the enhancement of keratin production in the trachea.

Squamous metaplasia is not observed in the small intestine, even in severe vitamin-A deficiency [7]; however, mucin biosynthesis, as measured by the incorporation of 3H-glucosamine, is decreased [8]. We were able to raise an antibody to the purified goblet cell glycoprotein [9]. Indirect immunofluorescence studies clearly indicated the presence of a cross-reactive antigen in a variety of rat epithelial tissues. Vitamin-A deficiency caused a marked drop in the amount of cross-reacting antigen in a variety of epithelial tissues, including the trachea [10]. Therefore, we can conclude that, in addition to cell division of mucous cells, vitamin A also controls mucus production.

Vitamin-A control of keratin gone expression

A later event during the progression of vitamin-A deficiency is keratin gene activation and consequent keratin production. A co-ordinated expression of acidic and basic keratins takes place in vitamin-A deficient hamster tracheas [11]. These keratins are not readily detectable in hamsters fed vitamin A or in tracheas cultured in the presence of retinoic acid. Notwithstanding the morphological similarity between the epidermis and the squamoid epidermoid tracheas, keratin gene products expressed in vitamin-A-deficient tracheas are not the same as in the skin. One outstanding difference is that keratin 1 (67,000 daltons), a prominent epidermal keratin, is not produced in vitamin-A-deficient tracheas [11].

Carcinogenesis

Exposure to chemical carcinogens, such as benzo[a]pyrene and 7,12-dimethylbenz[a]anthracene, causes similar squamous metaplastic lesions in the trachea [12]. When tracheas from four-week-old hamsters fed a normal diet were cultured in the presence of either benzo[a]pyrene or 7,12-dimethylbenz[a]anthracene for two weeks without retinoic acid, they developed squamoid metaplastic lesions. These lesions were not visible when retinoic acid was included in the medium containing the carcinogens (F. L. Huang et al., in preparation). Thus, clearly, retinoic acid is capable of repairing the squamoid metaplasia caused by carcinogen exposure. The squamoid lesions caused by the presence of the carcinogen were also positive by immunofluorescence with keratin antibodies (F. L. Huang et al., in preparation).

Importance of nutritional status in histogenesis: The concept of exotrophism

Organisms capable of synthesizing various essential nutrients are said to be prototrophic for those nutrients. Neurospora is an organism that is prototrophic for pantothenic acid, among other nutrients. When Neurospora is treated with a mutagenic dose of ultraviolet radiation, the result may be the establishment of a requirement for pantothenic acid in the offspring, which is said to have become a "pantothenic-acid auxotroph." Utilizing this approach, Beadle and Tatum [13] have elucidated the steps involved in the biosynthesis of various essential nutrients. Thus, auxotrophism is defined by the need for the exogenous supply of a nutrient in the presence of a mutated phenotype [14].

The mucociliary tracheas, or other epithelia, need vitamin A to maintain their differentiation. The tracheal squamoid cell, which prevails under conditions of vitamin-A deficiency, however, apparently does not need the vitamin. We propose to call this squamous cell "exotrophic" for vitamin A, that is, as having escaped the vitamin-A-requiring status normally characteristic of the columnar epithelium of the trachea. In the presence of vitamin A, this cell is replaced by the normal columnar cells. If, however, the supply of the vitamin is scarce, a conditional "vitamin-A exotroph," that is the squamoid cell, may persist at the site. This exotroph may become permanent by the action of a mutagenic agent, which would fix the exotrophic state, as shown schematically in figure 8. It is then possible to postulate that, when subject to the action of carcinogens and/or tumour-promoting substances, fixed exotrophic cells might divide and contribute to a tumour.

Figure 8

What is the advantage of the exotrophic state to the tumour cell? Quite simply that it has bypassed the requirement for the essential substance and thus the stringency of normal growth control. A cell can become more autonomous as it bypasses the requirements for more essential nutrients. The end result would be a cell that has been liberated from the usual constraints of regulatory substances and that may eventually prevail over other, normally regulated cells.

We and others have examined hepatoma cells and have found that, in general, they contain very little if any retinyl palmitate, whereas the surrounding hepatic host tissue contains normal concentrations of retinyl palmitate [15]. Transplanted hepatomas, whether minimally or maximally deviated from normal, are devoid of retinyl palmitate, whereas the host rat liver tissue and regenerating liver contain normal levels of the vitamin (table 2). Naturally, the lack of detectable retinyl palmitate in hepatoma tissue may be due to a variety of reasons, which are not mutually exclusive, among which are the following:

• The hepatoma cell may destroy vitamin A as it comes in contact with it, as suggested by the findings of Muto et al. [16] that some hepatomas may form anhydroretinol from retinol. - The deficiency may be the result of defective transport for retinol.
• A hepatic retinol exotroph may be the stem cell of hepatomas.

TABLE 2. Retinyl palmitate of hepatoma, host rat and regenerating liver postnuclear membranes (nanograms per milligram of protein)

	Liver	Tumour
20-1-1	580 ± 30	<1.6
16-2-1	513 ± 52	<1.6
7787-1-1	400 ± 21	<1.6
9618A-1-1	942 ± 2.2	<1.6
44-1-2	547 ± 21	<1.6
5123D-1-1	150 ± 3.8	<1.6
3924A-1-1	177 ± 59	<1.6
7800-1-1	73 ± 1.8	<1.6
5123 tc 1-2	363 ± 6.7	<1.6
7777-2-1	243 ± 38	<1.6
Primary cystic tumour	302 ±28	<1.6
Regenerating liver
24 hours	113 ± 7
48 hours	100 ±10

Summary and conclusions

In this paper we have suggested the new concept of exotrophic cells, i.e. cells that have conditionally escaped the need for an essential nutrient, such as vitamin A. These exotrophs might become fixed by a mutation and eventually contribute to the tumorigenic phenotype.

The discovery of the retinoic acid receptor (RAR) has opened up new horizons in the search for the mechanism of action of retinoic acid [17; 18]. It is intriguing that a second retinoic acid receptor, RARE, is abundantly expressed in hepatoma tissue and not in normal liver; Benbrook et al. [191 suggest that the erroneous expression of the RARE might contribute to tumour development in liver. How and whether these findings relate to the vitamin-A-deficient status of hepatoma cells remains to be understood.

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