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The Stimulation of Epidermal Keratinization by a Protein Isolated from the Submaxillary Gland of the Mouse*
Preliminary and Short Report THE STIMULATION OF EPIDERMAL KERATINIZATION BY A PROTEIN ISOLATED FROM THE SUBMAXILLARY GLAND OF THE MOUSE* STANLEY COHEN, PH.D.** AND GEORGE A. ELLIOTT, D.V.M. A heat-stable protein has recently been isolated from the submaxillary gland of the male mouse which, upon injection, elicits precocious eyelid
namely, an enhancement of epidermal keratiniza-
mouse and rat (1). The eyelids of the treated newborn mouse will separate as early as the sixth day after birth, whereas the eyelids of control animals
MATERIALS AND METHODS
tion and an increase in the overall thickness of the epidermis.
separation and tooth eruption in the newborn
The preparation of the biologically active protein has been described (1). From 5 to 7 mg of the pure protein was obtained from the submaxil-
require twelve to thirteen days to separate. The protein has a molecular weight of approximately 15,000 and has the unusual property of being
lary glands of 150 adult male mice (Swiss Webster).
110
\;- ) 79' if
FIG. la and b. Cross sections of the eyelid area from control, la, and experimental, lb, 8-day-old rats. The experimental animal had received daily injections (1 jg per 1 gm body weight) of the active protein. )< 100. devoid of lysine and phenylalanine. All of the Unless otherwise noted, the final pure protein was used in these experiments. The isolated protein other common amino acids are present. In this note, histological evidence is presented was dissolved in distilled water and daily subindicating that the observed eyelid separation is a cutaneous injections in the ventral neck region reflection of a more generalized biological effect, were made into the experimental animals. The quantities injected are given in the legends to the * From the Department of Biochemistry and the Figures. Litter mates receiving injections of Department of Pathology, Vanderbilt University distilled water were used as control animals. School of Medicine, Nashville, Tennessee. ** Research Career Development Awardee of At the termination of the injection period, desired specimens were removed, fixed in 10 per the United States Public Health Service. This investigation was supported in part by cent neutral formalin, decalcified when necessary, Research Grant RG-6638 from the National In- and embedded by standard procedures. Tissue stitutes of Health, United States Public Health sections were stained with routine hematoxylin Service. and eosin stain. Received for publication May 19, 1962. 1
2
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
.4t
:
a
S -
Fo. 2a and b. Sections of mid-dorsal back skin (epidermis) from control, 2a, and experimental, 2b, 12-day-old mice. The experimental animal had received daily injections (4 jg per 1 gm body weight) of the active protein. X 470. 0BSEavATI0NS AND DISCUSSION
intervals. The animals in each group showed a very similar histological picture.
In our first series of experiments five groups of Typical cross sections of the eyelid area from newborn mice (4 animals in each group) and five groups of newborn rats (3 animals in each group) experimental and control 8-day-old rats are shown received daily injections of the protein for periods in Figure la and lb. It can be seen that in the ranging from 1 to 20 days. A similar number of control animal the epidermis connecting the litter mates were used as control ammals and eyelids had just started to keratinize, whereas in received injection of distilled water. The groups the experimental animal the kcratinization process of animals were sacrificed at three to five day was considerably advanced and the eyelids had
STIMULATION OF EPIDERMAL KERATINIZATION
3
P. -
—e
a::-, —• tr—---. -s... ..s_. •-—— 1 •;
—
FIG. 3a and b. Sections of mid-dorsal back skin (epidermis) from control, 3a, and experimental, 3b, 12-day-old rats. The experimental animal had received daily injections (0.5 tg per 1 gm body weight) of the active protein. X 200.
begun to separate. An almost identical histological
picture was observed with the 8-day-old mice. Precocious separation of the eyelids was also noted by gross observation in experiments with newborn
and 2b are illustrated typical sections of middorsal back skin (epidermal portion) from 12-dayold experimental and control mice. It can be seen
that in the skin of the experimental animal the thickness of both the keratin and cellular layers The question of whether these keratinizing of the epidermis has increased. Mid-dorsal skin changes were limited to the eyelid area or were sections prepared from the 12-day-old rats indimore general was then examined. In Figure 2a cated that the skin of this species also responds rabbits and dogs.
4
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
FIG. 4a and b. Sections of the epidermis of the tails of control, 4a, and experimental, 4h, 45-day-old mice. The experimental animal had received daily injections (8 pg per 1 gm body weight) of a partially purified fraction (Sephadex 0-75 fraction as described in reference 1). The injections were started when the animals were 13 days old. X 200.
to the injection of the active protein. Figure 3a and 3b illustrate the results obtained after 12 daily injections of the protein into newborn rats.
animals. In Figure 4a and 4b are shown typical sections of the epidermis of the tail in such animals. In Figure Sn and 5b are illustrated sections of the epidermis taken from the plantar surface of the hind foot of these mice. It can be seen that in both areas there is a marked increase in the
Our work up to this point involved only the newborn animal. In another set of experiments twelve normal mice (six male and six female, Carworth Farms-i) ranging in age from 12 to 20 thickness of the epidermal layers. No such marked days received daily injections of a partially differences were noted in sections of mid-dorsal purified preparation of the factor. Again, a similar
number of litter mates were used as control
animals and received injections of distilled water. After 3 to 4 weeks of such injections, gross observa-
skin.
It should be mentioned that the injection of the purified protein into all of the newborn animals tested also results in a distinct inhibition in the general growth rate as measured by either body
tion of the mice revealed a clearly visible increase in the diameter of the tail in all of the experimental weight or body size (1). However, no inhibition of
STIMULATION OF EPIDERMAL KERATINIZATION
a— —
cE-r- :--
C
-
5
---C-'-.-- -! -
Fio. 5a and b. Sections of the epidermis taken from the plantar surface of the hind foot of control, 5a, and experimental, 5b, 39-day-old mice. The experimental animal had received daily injections (8 pg per 1 gm body weight) of the partially purified protein described in Figure 4a and b. The injections were started when the animals were 14 days old. X 200.
weight gain was observed in the mice when the maxillary gland protein. The questions of whether injections were started 12 to 20 days after birth. the effects of the isolated protein are limited to The reason for this difference is not known. epidermal structures and whether all epidermal There remain, of course, many unresolved structures are responsive are under study. problems. Among these are the role (if any) the In summary, the subcutaneous injection of a isolated protein plays in the normal differentiation protein isolated from the submaxillary gland of the of the epidermis and the mechanism by which the male mouse has been showed to enhance the protein stimulates the keratinizing process. It is keratinizing process in the epidermis of a variety not known whether the epidermis is affected of animal species.
directly or secondarily. It is hoped that tissue culture studies will help clarify this question. The
effects of a number of vitamins and hormones, such as vitamin A and estrogens, on epidermal structures has been reviewed recently by Laschet (2). The relationship between these various factors
REFERENCES 1. COnEN, S.: Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem., 237: 1555, 1962.
is obscure. In preliminary experiments in our 2. LASCHET, U.: Die Wirkung von Vitaminen und laboratory, large doses of vitamin A did not Hormonen auf das Epithel. Z. Vitamin. Hormon. Fermentforsch., 12: 1, 1961. inhibit the eyelid-opening effects of the sub-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.
namely, an enhancement of epidermal keratiniza-
mouse and rat (1). The eyelids of the treated newborn mouse will separate as early as the sixth day after birth, whereas the eyelids of control animals
MATERIALS AND METHODS
tion and an increase in the overall thickness of the epidermis.
separation and tooth eruption in the newborn
The preparation of the biologically active protein has been described (1). From 5 to 7 mg of the pure protein was obtained from the submaxil-
require twelve to thirteen days to separate. The protein has a molecular weight of approximately 15,000 and has the unusual property of being
lary glands of 150 adult male mice (Swiss Webster).
110
\;- ) 79' if
FIG. la and b. Cross sections of the eyelid area from control, la, and experimental, lb, 8-day-old rats. The experimental animal had received daily injections (1 jg per 1 gm body weight) of the active protein. )< 100. devoid of lysine and phenylalanine. All of the Unless otherwise noted, the final pure protein was used in these experiments. The isolated protein other common amino acids are present. In this note, histological evidence is presented was dissolved in distilled water and daily subindicating that the observed eyelid separation is a cutaneous injections in the ventral neck region reflection of a more generalized biological effect, were made into the experimental animals. The quantities injected are given in the legends to the * From the Department of Biochemistry and the Figures. Litter mates receiving injections of Department of Pathology, Vanderbilt University distilled water were used as control animals. School of Medicine, Nashville, Tennessee. ** Research Career Development Awardee of At the termination of the injection period, desired specimens were removed, fixed in 10 per the United States Public Health Service. This investigation was supported in part by cent neutral formalin, decalcified when necessary, Research Grant RG-6638 from the National In- and embedded by standard procedures. Tissue stitutes of Health, United States Public Health sections were stained with routine hematoxylin Service. and eosin stain. Received for publication May 19, 1962. 1
2
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
.4t
:
a
S -
Fo. 2a and b. Sections of mid-dorsal back skin (epidermis) from control, 2a, and experimental, 2b, 12-day-old mice. The experimental animal had received daily injections (4 jg per 1 gm body weight) of the active protein. X 470. 0BSEavATI0NS AND DISCUSSION
intervals. The animals in each group showed a very similar histological picture.
In our first series of experiments five groups of Typical cross sections of the eyelid area from newborn mice (4 animals in each group) and five groups of newborn rats (3 animals in each group) experimental and control 8-day-old rats are shown received daily injections of the protein for periods in Figure la and lb. It can be seen that in the ranging from 1 to 20 days. A similar number of control animal the epidermis connecting the litter mates were used as control ammals and eyelids had just started to keratinize, whereas in received injection of distilled water. The groups the experimental animal the kcratinization process of animals were sacrificed at three to five day was considerably advanced and the eyelids had
STIMULATION OF EPIDERMAL KERATINIZATION
3
P. -
—e
a::-, —• tr—---. -s... ..s_. •-—— 1 •;
—
FIG. 3a and b. Sections of mid-dorsal back skin (epidermis) from control, 3a, and experimental, 3b, 12-day-old rats. The experimental animal had received daily injections (0.5 tg per 1 gm body weight) of the active protein. X 200.
begun to separate. An almost identical histological
picture was observed with the 8-day-old mice. Precocious separation of the eyelids was also noted by gross observation in experiments with newborn
and 2b are illustrated typical sections of middorsal back skin (epidermal portion) from 12-dayold experimental and control mice. It can be seen
that in the skin of the experimental animal the thickness of both the keratin and cellular layers The question of whether these keratinizing of the epidermis has increased. Mid-dorsal skin changes were limited to the eyelid area or were sections prepared from the 12-day-old rats indimore general was then examined. In Figure 2a cated that the skin of this species also responds rabbits and dogs.
4
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
FIG. 4a and b. Sections of the epidermis of the tails of control, 4a, and experimental, 4h, 45-day-old mice. The experimental animal had received daily injections (8 pg per 1 gm body weight) of a partially purified fraction (Sephadex 0-75 fraction as described in reference 1). The injections were started when the animals were 13 days old. X 200.
to the injection of the active protein. Figure 3a and 3b illustrate the results obtained after 12 daily injections of the protein into newborn rats.
animals. In Figure 4a and 4b are shown typical sections of the epidermis of the tail in such animals. In Figure Sn and 5b are illustrated sections of the epidermis taken from the plantar surface of the hind foot of these mice. It can be seen that in both areas there is a marked increase in the
Our work up to this point involved only the newborn animal. In another set of experiments twelve normal mice (six male and six female, Carworth Farms-i) ranging in age from 12 to 20 thickness of the epidermal layers. No such marked days received daily injections of a partially differences were noted in sections of mid-dorsal purified preparation of the factor. Again, a similar
number of litter mates were used as control
animals and received injections of distilled water. After 3 to 4 weeks of such injections, gross observa-
skin.
It should be mentioned that the injection of the purified protein into all of the newborn animals tested also results in a distinct inhibition in the general growth rate as measured by either body
tion of the mice revealed a clearly visible increase in the diameter of the tail in all of the experimental weight or body size (1). However, no inhibition of
STIMULATION OF EPIDERMAL KERATINIZATION
a— —
cE-r- :--
C
-
5
---C-'-.-- -! -
Fio. 5a and b. Sections of the epidermis taken from the plantar surface of the hind foot of control, 5a, and experimental, 5b, 39-day-old mice. The experimental animal had received daily injections (8 pg per 1 gm body weight) of the partially purified protein described in Figure 4a and b. The injections were started when the animals were 14 days old. X 200.
weight gain was observed in the mice when the maxillary gland protein. The questions of whether injections were started 12 to 20 days after birth. the effects of the isolated protein are limited to The reason for this difference is not known. epidermal structures and whether all epidermal There remain, of course, many unresolved structures are responsive are under study. problems. Among these are the role (if any) the In summary, the subcutaneous injection of a isolated protein plays in the normal differentiation protein isolated from the submaxillary gland of the of the epidermis and the mechanism by which the male mouse has been showed to enhance the protein stimulates the keratinizing process. It is keratinizing process in the epidermis of a variety not known whether the epidermis is affected of animal species.
directly or secondarily. It is hoped that tissue culture studies will help clarify this question. The
effects of a number of vitamins and hormones, such as vitamin A and estrogens, on epidermal structures has been reviewed recently by Laschet (2). The relationship between these various factors
REFERENCES 1. COnEN, S.: Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the newborn animal. J. Biol. Chem., 237: 1555, 1962.
is obscure. In preliminary experiments in our 2. LASCHET, U.: Die Wirkung von Vitaminen und laboratory, large doses of vitamin A did not Hormonen auf das Epithel. Z. Vitamin. Hormon. Fermentforsch., 12: 1, 1961. inhibit the eyelid-opening effects of the sub-
THE JOURNAL OF INVESTIGATIVE DERMATOLOGY
94
linolenic acid extract. Arch. This pdf is a scanned copy UV of irradiated a printed document.
24. Wynn, C. H. and Iqbal, M.: Isolation of rat
skin lysosomes and a comparison with liver Path., 80: 91, 1965. and spleen lysosomes. Biochem. J., 98: lOP, 37. Nicolaides, N.: Lipids, membranes, and the 1966.
human epidermis, p. 511, The Epidermis
Eds., Montagna, W. and Lobitz, W. C. Acascopic localization of acid phosphatase in demic Press, New York. human epidermis. J. Invest. Derm., 46: 431, 38. Wills, E. D. and Wilkinson, A. E.: Release of 1966. enzymes from lysosomes by irradiation and 26. Rowden, C.: Ultrastructural studies of kerathe relation of lipid peroxide formation to tinized epithelia of the mouse. I. Combined enzyme release. Biochem. J., 99: 657, 1966. electron microscope and cytochemical study 39. Lane, N. I. and Novikoff, A. B.: Effects of of lysosomes in mouse epidermis and esoarginine deprivation, ultraviolet radiation and X-radiation on cultured KB cells. J. phageal epithelium. J. Invest. Derm., 49: 181, 25. Olson, R. L. and Nordquist, R. E.: Ultramicro-
No warranty is given about the accuracy of the copy.
Users should refer to the original published dermal cells. Nature, 216: 1031, 1967. version of1965. the material. vest. Derm., 45: 448, 28. Hall, J. H., Smith, J. G., Jr. and Burnett, S. 41. Daniels, F., Jr. and Johnson, B. E.: In prepa1967.
Cell Biol., 27: 603, 1965.
27. Prose, P. H., Sedlis, E. and Bigelow, M.: The 40. Fukuyama, K., Epstein, W. L. and Epstein, demonstration of lysosomes in the diseased J. H.: Effect of ultraviolet light on RNA skin of infants with infantile eczema. J. Inand protein synthesis in differentiated epi-
C.: The lysosome in contact dermatitis: A ration. histochemical study. J. Invest. Derm., 49: 42. Ito, M.: Histochemical investigations of Unna's oxygen and reduction areas by means of 590, 1967. 29. Pearse, A. C. E.: p. 882, Histochemistry Theoultraviolet irradiation, Studies on Melanin, retical and Applied, 2nd ed., Churchill, London, 1960.
30. Pearse, A. C. E.: p. 910, Histacheini.stry Thearetscal and Applied, 2nd ed., Churchill, London, 1960.
31. Daniels, F., Jr., Brophy, D. and Lobitz, W. C.: Histochemical responses of human skin fol-
lowing ultraviolet irradiation. J. Invest. Derm.,37: 351, 1961.
32. Bitensky, L.: The demonstration of lysosomes by the controlled temperature freezing section method. Quart. J. Micr. Sci., 103: 205, 1952.
33. Diengdoh, J. V.: The demonstration of lysosomes in mouse skin. Quart. J. Micr. Sci., 105: 73, 1964.
34. Jarret, A., Spearman, R. I. C. and Hardy, J. A.:
Tohoku, J. Exp. Med., 65: Supplement V, 10, 1957.
43. Bitcnsky, L.: Lysosomes in normal and pathological cells, pp. 362—375, Lysasames Eds., de Reuck, A. V. S. and Cameron, M. Churchill, London, 1953.
44. Janoff, A. and Zweifach, B. W.: Production of inflammatory changes in the microcirculation by cationic proteins extracted from lysosomes. J. Exp. Med., 120: 747, 1964.
45. Herion, J. C., Spitznagel, J. K., Walker, R. I. and Zeya, H. I.: Pyrogenicity of granulocyte lysosomes. Amer. J. Physiol., 211: 693, 1966.
46. Baden, H. P. and Pearlman, C.: The effect of ultraviolet light on protein and nucleic acid synthesis in the epidermis. J. Invest. Derm.,
Histochemistry of keratinization. Brit. J. 43: 71, 1964. Derm., 71: 277, 1959. 35. De Duve, C. and Wattiaux, R.: Functions of 47. Bullough, W. S. and Laurence, E. B.: Mitotic control by internal secretion: the role of lysosomes. Ann. Rev. Physiol., 28: 435, 1966. the chalone-adrenalin complex. Exp. Cell. 36. Waravdekar, V. S., Saclaw, L. D., Jones, W. A. and Kuhns, J. C.: Skin changes induced by
Res., 33: 176, 1964.