Leptin induces altered differentiation of keratinocytes by inducing insulin resistance: implications for metabolic syndrome-induced resistance of psoriatic therapy

Abstract

Background: Psoriatic patients tend to develop metabolic syndrome (MS). MS accelerates psoriasis, but the exact molecular mechanisms are poorly understood.

Objectives: We aim to investigate the impact of leptin on keratinocyte insulin sensitivity and explore its underlying molecular mechanism, which might play a role in the pathogenesis of this disease.

Methods: ELISA and immunohistochemistry were applied respectively to detect the level of leptin in serum and in lesion of psoriatic patients with and without MS. The HaCaT cell line was cultured and western-blot assay was performed to assess the change of insulin sensibility. q-PCR and western-blot assay were applied to detect the SOCS3 expressions. Knockdown of SOCS3 were generated in HaCaT cell line by siRNA. Leptin and insulin were treated for 6 days and K10 expression was evaluated by western-blot assay.

Results: Patients with MS had higher level of leptin in serum and lesions than their counterparts without MS. Serum levels of leptin was negatively correlated to PASI decline index in psoriatic patients. Long-term treatment of leptin induced insulin resistance in HaCaT cell line, as indicated by elevated expression of p-IRS-1 (ser636) and lower p-PKB (ser473). Leptin treatment up-regulated the mRNA and protein expression of SOCS3. Knockdown of SOCS3 blocked the effect of leptin-induced insulin resistance. Leptin treatment attenuated insulin-elicited K10 expression.

Conclusions: Leptin induces insulin resistance by upregulating SOCS3 and give rise to differentiation disorder of keratinocyte. Insulin resistance may serve as a target for anti-psoriatic therapies.

Keywords:

• Psoriasis
• metabolic syndrome
• leptin
• keratinocyte
• insulin resistance

1. Introduction

Psoriasis is a common skin disease. Its incidence rate stands at 1% among the world population and is still on the rise. The condition tends to recur, seriously affecting the patients’ physical and mental health and quality of life (1). Mounting evidence suggest that psoriasis is a systemic disease, rather than a skin disorder, and is complicated with obesity, dyslipidemia, hypertension, impaired glucose tolerance or diabetes and cardiovascular diseases (2). 10% to 30% of psoriasis patients have metabolic syndrome(MS), suggesting psoriasis bears a close relationship with MS (3). Our previous studies found that, 10 days after the same treatment, the PASI decline index was much lower in psoriasis patients with MS than in those without MS (4). So far, no effective therapy is available for MS-induced therapy resistance. Understanding the relationship between MS and psoriasis and the molecular mechanism is of great significance to the treatment of psoriasis patients with MS.

Leptin is an adipocytokine secreted by adipose tissue. The level of leptin is directly proportional to the total amount of systemic adipose tissue. Serum leptin is elevated in patients with MS. Several studies have demonstrated that leptin is associated with psoriasis (5). Leptin levels are increased in patients with psoriasis, and serum leptin level is positively correlated with the severity of psoriasis (6). Leptin induces altered differentiation of keratinocytes, thereby aggravating psoriasis (6). Taken together, the aforementioned results suggest that leptin acts as a key molecule in the development of resistance to treatment of psoriasis caused by MS. However, the exact mechanism by which leptin interferes with differentiation of keratinocytes remains unclear.

Insulin plays an important role in skin physiology. The insulin receptors and all downstream signaling pathway proteins are expressed in the epidermis and can be activated by insulin (7). In a mouse model with skin insulin receptor knocked down, keratinocyte proliferation and differentiation was seriously affected (8). Insulin receptor substrate 1(IRS-1) is a key molecule of the insulin signaling pathway. The skin of IRS-1 knockout mice was thinner and more transparent than that of normal mice, and the differentiation of epidermal cells is also affected, as indicated by a decreased K1 expression (9). The above results indicate that the insulin signaling pathway is important for maintaining the normal function of the epidermis. Similar to adipocytes and liver cells, keratinocytes also develop insulin resistance under certain conditions. Studies have found that insulin resistance exists in the epidermis of psoriatic lesions, and insulin resistance of keratinocytes interferes with the differentiation of keratinocytes (10).

Long-term stimulation with high level leptin can induce insulin resistance in liver cells. Leptin binds to the leptin receptor on the cell membrane and activates the JAK-STAT pathway. STAT entering the nucleus can cause SOCS3 expression. SOCS3 in combination with IRS-1 inhibits the activity of IRS-1 and ultimately degrades IRS-1 (11). These findings suggest that the leptin-induced differentiation disorder of keratinocytes might be mediated by insulin resistance. We found that leptin could cause insulin resistance in keratinocytes, thereby disrupting differentiation. Our results are important for further understanding of the relationship between MS and psoriasis, and provide a theoretical basis for using insulin resistance as a therapeutic target for psoriasis patients with MS.

2. Materials and methods

2.1. Chemicals and antibodies

All chemicals were purchased from Sigma (China), unless stated otherwise. Leptin was from Pepro Tech Inc. (Rocky Hill, NJ, USA) and phospho-specific and corresponding pan antibodies were from Cell Signaling Technology (Frankfurt, Germany). Actin antibody and keratin 10 were from Abcam (Cambridge, MA).

2.2. Immunohistochemistry

Biopsy specimens were collected from lesional skin of 10 psoriatic patients with MS and 10 psoriatic patients without MS before inception of therapy, and from healthy-looking skin of 3 healthy people. The sample collection began after written informed consent was obtained from the participants. The samples were cut into 8-mm cryosections and paraformaldehyde-fixed before staining with leptin antibody. Antibody staining intensities were evaluated by H-Score analysis as follows: no staining (0), mean (1), moderate (2), strong (3), and very strong (4). Semiquantitative H-Score values were calculated by counting positive staining cells in 5 randomly selected fields for each group [H-Score: ∑Pi (i + 1) (Pi: % positively stained cells; i: intensity of staining)]. This study has been approved by the local ethics committee of Chinese PLA General Hospital and all procedures were conducted in strict accordance with the Declaration of Helsinki.

2.3. Cell culture and conditions

The spontaneously immortalized human keratinocyte cell line (HaCaT) (Prof Fusenig, DKFZ, Heidelberg, Germany) was cultured in carbonate-buffered Hank’s medium with 10% fetal calf serum (Biochrom, Berlin, Germany), and 1% penicillin/streptomycin solution (Invitrogen, Karlsruhe, Germany) at 37 °C in 5% CO₂ atmosphere.

2.4. Western immunoblotting

Cells were lysed in lysis buffer (Cell Signaling Technology), normalized, subjected to SDS-PAGE, and blotted onto polyvinylidene difluoride membranes. After being blocked in 5% milk/Tris-buffered saline and Tween 20 (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20), membranes were probed with the indicated antibodies and visualized with horseradish peroxidase-conjugated secondary antibodies using LumiGlo reagent (Cell Signaling Technology).

2.5. Differentiation of keratinocytes

HaCaT cells were seeded into Ca2+-free medium and induced with insulin. The medium was changed every 48 h. Cells were harvested and either lysed and subjected to Western blotting or analyzed by flow cytometry. For the latter, cells were fixed with Cytofix (BD, Heidelberg, Germany), then permeabilized with Perm Buffer III (BD) and stained with phycoerythrinlabeled keratin 10 antibody. As an internal control, cells were stained with an allophycocyanin-labeled anti-actin antibody. For analysis, the geometric mean of unstained cells was subtracted from the geometric mean of stained cells of the same sample.

2.6. siRNA-mediated knockdown

Stealth siRNA directed against SOCS3 (50-CCAAAUUCUCCGAGGUCUAAAGUAU-30) and BLOCK-IT Negative Control were synthesized by Invitrogen. siRNAs were transfected using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. Briefly, 80 pmol siRNA duplexes (final concentration 66 nM) and 4 ml Lipofectamine 2000 were diluted separately in OptiMEM-I (Invitrogen), mixed, and added to the cells.

3. Results

3.1. Leptin serves a critical role in the development of therapeutic resistance in psoriatic patients with MS

Our previous studies found that leptin was elevated in the epidermis of psoriasis patients (6). We further compared the leptin levels in lesional skin and serum in psoriatic patients with or without MS. We harvested skin tissues of normal human subjects (n = 3) and psoriatic lesions of patients without MS (n = 10) and those with MS (n = 10). Immunohistochemical staining showed that leptin was expressed only in the basal layer of healthy skin tissues and across the entire layers of epidermis of psoriasis patients (Figure 1A), and the expression was much stronger in lesional tissues with MS in terms of H-scores (p < 0.05, figure 1B). ELISA revealed that serum leptin levels were significantly higher in psoriatic patients with MS(n = 36) than in their counterparts without MS (n = 34, p < 0.05, figure 1C). We also found the serum leptin level was negatively correlated with the PASI score reduction index 10 days after NB-UVB therapy (Figure 1D n = 62 r =-0.4022, p = 0.0046).

3.2. Leptin induces insulin resistance of keratinocytes via SOCS3

Adipocytokines, such as TNF-α, IL-6 and leptin, are able to confer insulin resistance in metabolically-active tissues (12). Therefore, we examined whether leptin could confer similar effects in keratinocytes. After 72 h of leptin treatment, followed by insulin stimulation in HaCaT, we observed a rise in protein insulin receptor substrates 1(IRS-1)S636 phosphorylation and a reduction in protein kinase B (PKB) S473 phosphorylation, both being the indicators of insulin signaling inhibition (Figure 2A).

Figure 1. Leptin in the epidermis and serum are higher in psoriatic patients with MS.

(A) Skin sections of normal human skin (healthy) or psoriasis without MS (without MS) and with MS lesional (with MS) skins were immunohistochemically stained with leptin antibody.

(B) The intensity of immunohistochemical staining was quantitatively rated by H-score.

(C) Serum levels of leptin in psoriatic patients without MS or those with MS were detected by ELISA.

(D) The decrease rate of PASI of psoriatic patients treated by NB-UVB for 10 days was negatively correlated with the serum leptin level (Pearson’s correlation analysis, r = −0.4022, p < 0.01, n = 62).

Figure 2. Induction leptin-induces insulin resistance in keratinocytes viaed by SOCS3.

(A) Starved HaCaT cells were treated with 1 μg/ml leptin for 72 hours, followed by 10 minutes of stimulation with 100 nM insulin. Immunoblotting was performed with the indicated phospho-specific or total protein antibodies.

(B) Starved HaCaT cells were treated with 1 μg/ml leptin for 48 hours and the mRNA expression of SOCS3 was determined by q-PCR.

(C) Cells were treated with 1 μg/ml leptin for 48 or 72 hours and the protein expression of SOCS3 was detected by western-blotting.

(D) HaCaT cells were transfected with 80 pmol small interfering RNA (siRNA), serum-starved, and stimulated with 1 μg/ml leptin for 72 hours, followed by 10 minutes of stimulation with 100 nM insulin. Western-blotting was conducted with the indicated phospho-specific or total protein antibodies. PKB, rotein kinase B; P-PKB, phosphorylated PKB; IRS1, insulin receptor substrates.

Figure 3. Leptin-induced insulin resistance interferes with the differentiation of keratinocytes. HaCaT cells were treated with 100 nM insulin, 100 ng/ml and 1 μg/ml leptin for 6 days, and the expression of keratin 10 (K10) and actin was assessed by Western blotting.

Suppressor of cytokine signaling 3 (SOCS3) reportedly could phosphorylate IRS-1, as a negative feedback mechanism of insulin signaling, and leptin was found to induce expression of SOCS3 (13). A question naturally presents itself: Does SOCS3 mediate leptin-induced insulin resistance in keratinocytes? Therefore, we investigated the expression of SOCS3 in HaCaT cells treated with 1 μg/ml leptin for 48 or 72 h. Both mRNA (Figure 2B) and protein (Figure 2C) of SOCS3 were overexpressed. Then we blocked SOCS3 by small interfering RNA (siRNA)-mediated knockdown and found insulin-dependent PKB phosphorylation was partially restored in HaCaT cells (Figure 2D). These results indicated that SOCS3 is involved in the mediation of leptin-induced insulin resistance in keratinocytes.

3.3. Leptin interferes with the differentiation of keratinocytes by inducing insulin resistance

As one of hallmarks of psoriasis, altered differentiation of kerotinocytes is induced by insulin resistance in HaCaT and NHK cells. Our previous studies indicated that leptin leads to altered differentiation (6). We hypothesized that whether leptin could also inhibit insulin-dependent differentiation. We found that leptin blocked insulin-driven differentiation, as demonstrated by the down-regulated expression of keratin 10 (Figure 3). In summary, leptin worked by inducing keratinocytes insulin resistance, and thus blocking insulin-mediated differentiation.

4. Conclusion

The major finding of this study was that leptin, an adipokine, induced insulin resistance in keratinocytes, which, in turn, interfered with their differentiation. Increasing evidence exhibits that leptin plays an important role in the sustenance of psoriasic condition (5). So far, little is known about the exact mechanism by which leptin induces altered differentiation of keratinocytes. To our knowledge, no previous studies report that leptin could induce insulin resistance in keratinocytes, that this induction important for the development of psoriasis.

Leptin was found to affect the sensitivity of cells to insulin in a variety of cells, but the results were inconsistent. Some studies found that leptin could increase insulin sensitivity in liver, adipose tissues and striated muscles. In all these studies, the time of leptin stimulation was short (14). In other studies, cells were chronically stimulated by leptin, and the results were in stark contrast to findings with the short-term stimulation. In nerve cells, short-term leptin stimulation increases insulin sensitivity. When leptin stimulation time is prolonged, insulin sensitivity is reduced (15). In murine models, short-term stimulation with leptin caused an increase in AKT phosphorylation downstream of the insulin signaling pathway, as manifested by increased insulin sensitivity; while 14 days after leptin stimulation, AKT phosphorylation levels decreased, which was indicative of decreased insulin sensitivity (16). Thus, the effect of leptin on insulin sensitivity may be related to the length of leptin stimulation. This is consistent with our findings that 72 h after treatment of keratinocytes with leptin, the cells developed insulin resistance.

Having clarified that leptin could elicit insulin resistance in keratinocytes, we further explored the molecular mechanism of this effect. Our results indicated that leptin stimulation up-regulated SOCS3 expression in keratinocytes. Leptin stimulation was unable to induce insulin resistance in keratinocytes upon blockade of SOCS3 expression by siRNA. In hepatocytes, leptin bound to the leptin receptor on the cell membrane and activated the JAK-STAT pathway and STAT entering the nucleus induced SOCS3 expression. SOCS3 in combination with IRS-1 inhibited the activity of IRS-1 and ultimately degraded IRS-1 (11). Our results confirmed that up-regulated SOCS3 was associated with inhibited phosphorylation of IRS-1, but IRS-1 expression experienced no changes. This might be ascribed to the fact that hepatocytes are a kind of metabolism-related cells, and the molecules in signaling pathways involved in glucose metabolism are more sensitive.

We found that leptin could block the promoting effect of insulin on the differentiation of keratinocytes. Insulin could promote the differentiation of keratinocytes; IL-1β could induce keratinocytes to develop insulin resistance and prevent insulin from inducing keratinocyte differentiation (10). Our study indicated that leptin-induced insulin resistance exerted the same effect on keratinocytes. Given that altered differentiation of keratinocyte is one of the important features of psoriasis, our results suggest that leptin can aggravate psoriasis by interfering with the differentiation of keratinocytes by inducing insulin resistance.

Our previous study found that psoriatic patients with MS tend to be refractory to the treatment for psoriasis (6). To our knowledge, it was the first time that serum level of leptin was found to be negatively associated to the therapeutic effect on psoriasis. We compared the leptin in the lesions of psoriatic patients with MS and those without. We found that the level of leptin in psoriatic lesions was significantly higher in psoriatic patients with MS than in their counterparts without MS. The finding suggests that leptin might be a key molecule responsible for the MS-related poor response of psoriasis. However, it is necessary to acknowledge the limitations of this work. The sample size in our study was limited due to the requirement of obtaining consent for skin-tissue sampling. A larger sample size is needed to further confirm our study in the future.

On the basis of our observations and other studies, we hereby proposed a model: Metabolic syndrome is a chronic inflammatory state that produces a large number of inflammatory factors in the circulation, including the adipocytokine leptin. Leptin can enter the epidermis to stimulate keratinocytes on long-term basis and as a consequence, the cells develop insulin resistance, which hinders the normal differentiation of cells, exacerbates psoriasis and diminishes the therapeutic effect on psoriasis.

Studies demonstrated that dieting or combination use of insulin sensitizers could improve the efficacy of psoriasis treatment (17). Our study provided an experimental and theoretical basis for this finding. The reduction of leptin levels can ameliorate metabolic disorders and enhance insulin sensitivity in the body (18). Feasible treatment strategies to reduce the effects of metabolic syndrome on psoriasis therapy might include reducing leptin level, inhibiting the action of leptin on keratinocytes and reducing insulin resistance in keratinocytes.

Acknowledgements

We thank all the patients who agreed to take part in our study and all the doctors and nurses of the Inpatient Department of Dermatology of Chinese PLA general Hospital for their contributions and support to the research.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This work was supported by the [the Beijing Natural Science Foundation of China under Grant [number 7224344].