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Department of Pathology, Sackler School of Medicine, Tel Aviv University (E.W., N.S., M.T., S.N.-M., G.S.), Tel Aviv 69978, Israel; Faculty of Life Sciences, Bar Ilan University (M.G., T.T.), Ramat Gan 52900, Israel; Columbia University/Berrie Research Pavilion (D.A.), New York, New York 10032; and Department of Pathology, E. Wolfson Medical Center (I.A., S.N.-M.), Holon 58100, Israel
Address all correspondence and requests for reprints to: Efrat Wertheimer, M.D. Ph.D., Department of Pathology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel. E-mail: effy{at}patholog tau.ac.il.
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Abstract |
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Introduction |
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Several pathogenic mechanisms have been suggested to be involved in the development of diabetic complications (6, 7, 8, 9, 10, 11, 12); these include diabetic microangiopathy, premature aging of the skin fibroblasts, immune-mediated processes, changes in basement membrane structure and function, and others. However, the exact mechanism causing the various pathological conditions in skin is not known.
It is usually assumed that most diabetes complications are induced due to elevated glucose levels associated with this disease. Nevertheless, the diabetic milieu includes, in addition to hyperglycemia, abnormal insulin signaling. One could propose that if indeed insulin has a direct role in the normal function of skin, abnormal insulin signaling might take a part in the development of these complications as well. Little is known, however, about the roles of the insulin receptor (IR) and insulin signaling in normal skin physiology.
To investigate this question we recently studied a model system of skin keratinocytes in culture (13). In this model system, proliferation and differentiation of the cultured keratinocytes can be controlled by regulating the Ca2+ concentration in the growth medium (14). Using this model we previously demonstrated that the IR and insulin-like growth factor I (IGF-I) receptor (IGFR) are expressed in skin keratinocytes, and can be stimulated by insulin and IGF-I, resulting in the activation of an intracellular signaling pathway (13). Furthermore, we have shown that the activity level of both receptors is differentiation dependent (13).
In the present study we extended these experiments to identify the exact roles of insulin and IGF-I in skin keratinocytes. We studied the skin of IR knockout (IR-KO) transgenic mice in which the IR gene was inactivated by genetic manipulation (15). We were able to demonstrate, using skin sections as well as isolated keratinocytes, that the lack of IR expression leads to abnormal differentiation of skin keratinocytes, and that insulin-induced glucose transport was abrogated in IR null cells. These results indicate that IR is directly involved in the regulation of skin differentiation and glucose transport, and that abnormal insulin signaling might contribute to abnormalities in these processes.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell culture and protein lysate preparation
Primary mouse keratinocytes were prepared and maintained as
described previously (13). Briefly, freshly isolated
keratinocytes were cultured in Eagle’s medium (Biological Industries,
Beit Haemek, Israel) with 10% chelexed FCS (Biological
Industries), 1% antibiotics, and a Ca2+
concentration adjusted to 0.05 mM as previously described
(13). After 5 days in culture, to induce differentiation
the growth medium was switched to medium containing
Ca2+ at defined concentrations [0.05
mM (L), 0.12 mM (M), and 1.0 mM (H)
Ca2+] for 48 h. After 48 h, unless
indicated, cells were harvested by scraping into lysis buffer (PBS
containing 1% Triton X-100, 1 mM EDTA, 10 mM
sodium fluoride, 200 µM sodium orthovanadate, and a
protease inhibitor cocktail). The lysate was spun down at maximum speed
in a microcentrifuge, and the Triton-soluble supernatant was further
analyzed by SDS-PAGE and immunoblotting. The Triton-insoluble pellet
was kept for analysis of cytoskeletal proteins, as described below.
Protein concentrations were measured using a modified Lowry assay (DC
Protein Assay Kit, Bio-Rad Laboratories, Inc., Hercules,
CA).
Preparation of cytoskeletal protein samples for analysis of keratin
expression
The Triton-insoluble fraction (pellet), obtained as described
above, was incubated for 30 min in a special lysis buffer containing
-mercaptoethanol (20%) and SDS (5%). The samples were centrifuged
for 30 min at maximal speed in a microcentrifuge, and the lysate was
further analyzed by SDS-PAGE and Western blot analysis.
Immunoblotting analysis
The protein samples were analyzed by SDS-PAGE and transferred by
electroblotting onto nitrocellulose membranes. Immunoblotting was
performed as described previously (16) using rabbit
polyclonal antibodies against the IR, IGFR, insulin receptor
substrate-1 (IRS1), phosphoinositol 3-kinase (p85a), Shc,
and Crk (Santa Cruz Biotechnology, Inc., Santa Cruz, CA),
keratin 1 and 10 (gifts from Dr. S. H. Yuspa, NCI, NIH), GLUT1 (a
gift from Dr. S. Cushman, NIDDK, NIH), or monoclonal antibodies
recognizing phosphotyrosine residues (Upstate Biotechnology, Inc.; Lake Placid, NY). Filters were then incubated with the
appropriate secondary horseradish peroxidase-conjugated antirabbit
(Bio-Rad Laboratories, Inc.) or antimouse (Amersham Pharmacia Biotech, Piscataway, NJ) antibodies. Proteins were
detected using an enhanced chemiluminescence protein detection kit
(SuperSignal, Pierce Chemical Co., Rockford, IL).
Ligand-stimulated receptor phosphorylation in intact cells and
immunoprecipitation
Confluent cultures were incubated in the presence or absence of
10-6 M insulin
(Eli Lilly & Co., Indianapolis, IN) or
10-7 M IGF-I
(CytoLab, Rehovot, Israel) for 3 min at room temperature. The
reaction was terminated by aspiration of the incubation medium followed
by quick washes with ice-cold PBS on ice. Cells were lysed in cold
lysis buffer (PBS containing 1% Triton X-100, 1 mM EDTA,
10 mM sodium fluoride, 200 µM sodium
orthovanadate, and a protease inhibitor cocktail). Receptors were
immunoprecipitated with antiphosphotyrosine antibodies (Upstate Biotechnology, Inc.), followed by protein A/G-agarose beads
(Santa Cruz Biotechnology, Inc.) for 16–20 h at 4 C. The
beads were then washed twice with cold lysis buffer. The
immunoprecipitates were run on a SDS-PAGE, and
phosphotyrosine-containing proteins were detected by Western blotting
using appropriate antibodies, as described above.
2-Deoxy-[3H]glucose (dGlc) uptake
Glucose transport was evaluated by measuring dGlc according to
the method described previously (17). Briefly, on the day
of the experiment, cells were washed three times with PBS. Then, 0.1
mM 2-deoxyglucose/PBS with tracer amounts of dGlc (1
µCi/plate; ARC, St. Louis, MO) was added to the cells. Uptake was
allowed to continue for 10 min at room temperature. The reaction was
stopped by three quick washes with 1.0 ml cold PBS on ice, and cells
were lysed in 1% Triton X-100. The samples were counted in the
3H window of a Tri-Carb scintillation counter.
Uptake was linear under these conditions for up to 15 min (data not
shown). Mean values were determined from measurements of triplicate
samples under each experimental condition for each experiment.
Thymidine incorporation
Cell proliferation was evaluated by measuring
[3H]thymidine incorporation into DNA. Cells
were pulsed with [3H]thymidine (1 µCi/ml;
ICN, Irvine, CA) for 1 h at 37 C. After incubation, cells were
washed three times with PBS, incubated for 15 min at room temperature
in 5% trichloroacetic acid, and solubilized in 1% Triton X-100. The
radioactivity incorporated into the cells was counted in the
3H window of a Tri-Carb liquid scintillation
counter. Mean values were determined from measurements of triplicate
samples under each experimental condition for each experiment.
5'-Bromodeoxyuridine (BrdU) labeling and detection
Transgenic mice and control littermates were injected ip with
BrdU (Sigma, St. Louis, MO; 250 mg/kg in 0.9% NaCl). Mice
were killed 3 h after BrdU injection. Dorsal skin was embedded in
OCT (Miles, Elkhart, IN), fresh-frozen, and sectioned. Sections were
stained for BrdU by direct immunofluorescence, as described previously
(18), using an FITC- conjugated anti-BrdU antibody
(Becton Dickinson and Co., San Jose, CA). Briefly, slides
were incubated overnight at 4 C with the anti BrdU antibody. After
incubation slides were washed twice for 10 min each time with PBS for
10 min and mounted with glycerol buffer containing 1%
p-phenylenediamine (Sigma). Fluorescence was
examined by laser scanning confocal imaging microscopy (MRC1024,
Bio-Rad Laboratories, Inc., Hemel-Hempsted, UK).
Epidermal BrdU incorporation was quantitated by counting BrdU-labeled
nuclei in basal interfollicular epidermal cells from multiple randomly
chosen fields in BrdU-stained sections of skin.
Immunohistochemistry
Immunohistochemical analysis of keratin expression was carried
out using monospecific polyclonal antibodies to keratins as previously
described (18). Briefly, dorsal skin from IR-null mice and
control littermates was fresh-frozen and sectioned. Sections were
incubated overnight with appropriate antibodies against keratins 14, 1,
10, and 6 and against loricrin (all gifts from Dr. S. H. Yuspa,
NCI, NIH). Sections were then incubated with biotinylated secondary
antibody (Jackson ImmunoResearch Laboratories, Inc., West
Grove, PA), followed by incubation with horseradish
peroxidase-conjugated avidin-biotin complex reagent (Vector Laboratories, Inc., Burlingame, CA) with diaminobenzidine as the
substrate (Sigma, St. Louis, MO) and a hematoxylin
counterstain.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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skin of the IR-KO mouse appeared normal compared with the WT skin, the
epidermis and the dermis were of normal thickness, and the epidermis
contained the expected number of cellular layers in each epidermal
compartment. In addition, the development of hair follicles and their
structure was normal compared with that of WT skin.
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, the number of proliferating cells in
both the dermis and epidermis was significantly lower in IR-KO mice. At
higher magnification (Fig. 2, C and D) it can be seen that in both
IR-WT and IR-KO skin the proliferating cells in the epidermis are
restricted, as expected, to the basal layer, lying just above the
basement membrane. At this higher magnification it is easy to
appreciate the decreased number of proliferating cells in the epidermis
of IR-KO skin compared with the epidermis of IR-WT skin. In addition,
morphometric analysis was performed (18) by counting
labeled cells in multiple, randomly selected fields, and expressing
their incidence per epidermal length in each section. This analysis
gives a quantitative summary of the data, showing that in the IR-KO
skin proliferation is reduced by 40% (7.2 vs. 5.2
cells/arbitrary length unit, respectively, in IR-WT and IR-KO skin
sections).
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) the localization and intensity of the
immunohistochemical staining of most differentiation markers
representative of the various differentiation stages is similar in null
and wild-type skin. In both IR-WT and IR-KO mice the expression of
differentiation-specific keratins K1 and K10 is restricted to the
suprabasal layer of the epidermis, and loricrin was found to be
expressed in the granular compartment. The main change was seen in the
localization of K6 expression. In normal skin, K6 expression is
restricted to the hair follicles, and it is only transiently expressed
in the proliferating epidermis of wounds (20, 21, 22). As can
be seen, in WT skin the expression of K6 was indeed restricted to the
hair follicles. In contrast, in IR-KO epidermis K6 was widely expressed
in interfollicular epidermis in suprabasal and also some basal
cells.
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), there was no expression, as expected,
of the IR in skin keratinocytes isolated from IR-KO mice. The
expression levels of IGFR, IRS1, phosphoinositol 3-kinase
(p85a), Shc, and Crk were next examined. As shown (Fig. 4), all of
these proteins were expressed similarly in the WT skin keratinocytes
and the IR-KO mice regardless of the level of IR expression.
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). As can be seen, the wild-type nonimmortalized keratinocytes
exhausted their replication capacity after a fixed number of cycles
in vitro. In comparison, it seemed that there was a larger
decrease in the proliferative rate of the IR-KO cells at this stage.
However, although this change was repetitive, it was not statistically
significant.
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, increasing the Ca2+ to above 0.1
mM resulted in the wild-type cells in an
increased expression of K1 and K10, the earliest differentiation
markers, and later the induction of differentiation was associated with
the expression of loricrin and
filaggrin. In contrast,
keratinocytes from mice lacking IR expression exhibited an abnormal
differentiation process. As shown in Fig. 6, the progress of differentiation,
evaluated by following the expected induction of K1 and K10 expression,
was delayed in cells isolated from IR-KO mice compared with cells
isolated from WT-IR littermates. There was no significant change in
other markers examined (Fig. 6).
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and as was previously shown by us
(13), induction of differentiation is associated with a
decrease in IGFR autophosphorylation. In addition, it can be seen
that both insulin and IGF-I stimulation result in autophosphorylation
of IR and IGFR in WT cells (Fig. 7B). However, lack of IR expression
resulted in an a statistically significant increase in IGF-I-induced
IGFR autophosphorylation in proliferating as well as in differentiating
IR-KO keratinocytes (Fig. 7, A–C), demonstrating that activation of
the IGFR is elevated in compensation for the lack of IR signaling. In
contrast, there was no compensatory increase in IGFR
autophosphorylation when cells were stimulated by insulin (Fig. 7B).
Interestingly, the phosphorylation of IRS1 was inhibited by 30%, on
the average (Fig. 7B). Thus, the increase in IGFR autophosphorylation
cannot compensate for the lack of IR-induced IRS1 phosphorylation.
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). We
next examined the glucose transport rate using dGlc (Fig. 8B).
Proliferating skin keratinocytes isolated from WT-IR and IR-KO mice had
similar basal dGlc uptake of 66.7 and 63.3 dpm/µg protein·min,
respectively. Acute insulin stimulation did not affect the dGlc uptake
rate of either cell type (data not shown). On the other hand, chronic
insulin exposure increased the glucose transport rate of the WT cells,
whereas it was unchanged in IR-KO cells. Under similar conditions
elevation of the glucose transport rate into the cells in response to
chronic IGF-I stimulation was similar in WT and IR-KO mice (Fig. 8B).
These results suggest that the effects of insulin on glucose transport,
attributed by most researchers to the cross-activation of the IGFR by
insulin, are actually transmitted via insulin-induced activation of the
IR per se.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that in skin keratinocytes, insulin and IGF-I have different roles in skin proliferation and differentiation that are mediated via distinct signaling pathways (13, 19). Involvement of insulin in cellular proliferation and differentiation was shown in other tissues as well, including 3T3-L1 preadipocytes and C2 muscle cells (24, 25). A striking similarity can be found when comparing our results to those found in gastrointestinal enterocytes (26). IGFR and secreted IGF-I appear to be characteristic of proliferating cryptic cells, whereas differentiating cells show higher insulin binding and no secretion of IGF-I. However, this is the first time that a direct link has been demonstrated between IR expression/signaling and the cellular processes of proliferation and differentiation. Interestingly, the lack of IR was not associated with impairment of the adipocyte differentiation process, demonstrating a tissue-specific involvement of insulin and IGF-I in these processes (27).
Our studies indicate differences between the proliferation process in vivo and that of cultured skin keratinocytes in vitro. There was a marked decrease in the proliferation of basal keratinocytes in IR-KO skin compared with IR-WT skin. In contrast, there was not a statistically significant difference in the thymidine proliferation rate between cultured IR-KO and IR-WT cells. We postulate that this difference results from the effects of the diabetes milieu developing in the IR-KO mouse on the proliferation process, especially to the hyperglycemia developing in the IR-KO pups a few hours after birth. Indeed, in recent studies we found (unpublished results) that incubating skin keratinocytes in high glucose for several days results in a decrease in cell proliferation compared with that of cells incubated in physiological levels of glucose.
Differences in the differentiation of IR-KO keratinocytes in
vivo vs. that of cells cultured and induced to
differentiate in vitro were also found. The differentiation
process of intact skin seemed to be slightly impaired, whereas it is
significantly altered in cultured keratinocytes of the same animal. The
main change observed in the IR-KO skin was an increase in K6 expression
in the IR-KO epidermis compared with the IR-WT skin. In normal murine
and human epidermis, K6 is expressed constitutively in a variety of
internal stratified epithelia as well as in palmoplantar epidermis and
specialized cells of the hair follicle (20). K6 expression
was also shown to be induced during wound healing, skin disorders such
as psoriasis, skin tumors, and in response to the topical applications
of retinoic acid and phorbol esters (21, 22, 28). It was
initially thought that K6 expression was associated with
hyperproliferative conditions; however, it has been more recently
demonstrated that K6 is induced whenever the normal biology of
keratinocytes is disturbed (29, 30, 31). In the IR-KO
epidermis, the increase in K6 expression was associated with suppressed
keratinocyte proliferation. Such a case was shown in transforming
growth factor--overexpressing transgenic mice (32), in
which K6 was highly expressed, and keratinocyte proliferation was
suppressed. It was recently shown that K6 null mice exhibited severe
blistering and neonatal lethality (33), demonstrating that
K6 has an important role in skin and possibly in other organs as well.
However, the functional significance of increased expression of K6 on
keratinocyte biology under various conditions is as yet unknown. In
cultured keratinocytes, on the other hand, K6 expression is
constitutively elevated, probably due to the destruction of the normal
skin structure and cell to cell contacts (14). There was
no other change in the expression or localization of the other
differentiation markers tested in the skin samples derived from IR-KO
mice, in contrast to the abnormally reduced expression of K1 and K10 in
the differentiated cultured keratinocytes. The discrepancy probably
results from the short life span of the IR-KO mice. These mice die of
ketoacidosis within 3–5 days of birth (15). Indeed, it is
possible that other abnormalities would have developed later in life if
this phenotype was not lethal. Support for this possibility can be
found in studies showing (34) that even though the skin of
IR-KO mice is normal in appearance, the epidermis of the IGFR/IR double
nullizygous mouse is thinner than that of the IGFR single mutant.
Another explanation for the fewer changes seen in intact skin could be
that in the intact animal a compensatory mechanism is activated, which
does not exist in the cultured keratinocytes. For example, such a
mechanism could involve a process dependent, partially or fully, on the
dermal fibroblasts. It is known that local IGF-I and IGF-binding
proteins play a role in regulation of IGFR activity
(35, 36, 37). In addition, IGF-2 was shown to participate in
growth-promoting interactions with the IR during development
(34). Such IGF regulatory factors could be secreted not
only by the keratinocytes, but also by the dermal fibroblasts; their
absence in cell culture could lead to further or facilitated
deterioration of cellular physiology.
In the study we have also demonstrated that IGFR activity is increased in compensation for the lack of IR. It is generally accepted that the main role of insulin is regulation of glucose transport and metabolism, whereas IGF-I is involved in cellular growth and differentiation. However, it has been shown that IGF-I can affect glucose transport, and conversely, insulin may affect proliferation and differentiation (38, 39). Furthermore, functionally important IR-IGFR hybrids have been demonstrated on the cell surface (40, 41, 42). Finally, it is known that insulin and IGF-I can cross-activate IGFR and IR, respectively (38, 39). All of the above phenomena were shown to occur in cultured murine keratinocytes (13, 23) and could be used by the cells to induce compensatory activity of IGF-I or the IGFR in the absence of IR. Physiological support for the compensatory role of IGFR in the absence of IR can be found in a recent study showing that IGF-I caused a rapid and sustained decrease in plasma glucose in IR-KO mice. It was suggested that this effect was due to IGF-I stimulation of glucose uptake in skeletal muscle, although this hypothesis was not proven (43). Nonetheless, we have recently shown that in cultured IR-KO muscle cells, there is a compensatory increase in both the expression as well as the activity of the IGFR (44). Theoretically, this compensatory increase could allow the cells and tissues to develop to a certain point, as was suggested in leprechaun patients (45, 46). However, it is clear that IGFR overactivation was not able to replace all of the activities of insulin, as can be deduced from the severe phenotype of the leprechaun patients and the IR-KO mice. In addition, we found that IRS1 phosphorylation was not increased, but, rather, was inhibited in the IR null skin keratinocytes. Furthermore, although we have demonstrated that insulin can autophosphorylate the IGFR, insulin-induced glucose transport still requires the IR and is abolished in IR-KO cells. One could further suggest that as insulin and IGF-I play different roles in keratinocyte proliferation and differentiation, overactivation of the IGFR could by itself lead to some of the abnormalities seen in the IR-KO mice.
In conclusion, we demonstrated that insulin plays an active role in skin physiology. Indeed, it is plausible that states of insulin resistance such as diabetes might result in abnormal skin function, leading to impaired wound healing. It is hoped that further identification of the roles of insulin and IGF-I in skin will lead us to a better understanding of the pathogenesis of diabetic skin complications and possibly to the development of treatments and prophylaxis against these complications.
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Footnotes |
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2 Recipient of a Career Development Award from the Juvenile Diabetes
Foundation International and of the APF Kass Fund Award for Medical
Research.
Received March 31, 2000.
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| Endocrinology |
, WT;
, IR-KO) was carried out as detailed in A. Statistical analysis of
the results was performed by scanning densitometry. Values shown are
the mean ± SD of determinations from three separate
independent experiments (P < 0.01).