HIF - 1 α may promote glycolysis in psoriasis vulgaris via upregulation of CD147 and GLUT1

Abstract

Objectives: To analyze the expressions and distributions of hypoxia-inducible factor-1α (HIF-1α), CD147, and glucose transporter 1 (GLUT1) in epidermis from psoriasis vulgaris and normal people, and to explore the associations among these proteins and their roles in hypoxic HaCaT cell line.

Methods: The expression levels of HIF-1α, CD147, and GLUT1 were determined by immunohistochemistry staining in skin biopsies from 48 psoriasis vularis patients and 33 healthy subjects. Cobalt chloride (CoCl₂) was added into the culture media of HaCaT cells to mimic hypoxia while RNA interference and transfection technologies were used to explore the association among these proteins by quantitative real-time polymerase chain reaction and Western blotting. Glycolytic capacity was detected by ATP and lactate measurements.

Results: HIF-1α, CD147, and GLUT1 were highly expressed and the glycolytic capacity was increased in lesions of psoriasis vulgaris; HIF-1α upregulated the expression of CD147 and GLUT1, increased the lactate production and decreased the ATP level in CoCl₂-treated HaCaT cells, while CD147 and GLUT1 directly or indirectly bound to each other.

Conclusions: Glycolytic capacity increases in the injured keratinocytes of psoriasis vulgaris, suggesting that HIF-1α, CD147, and GLUT1 are associated with glycolysis, which can be considered as the promising targets for psoriasis therapy.

Keywords: CD147; glucose transporter 1; glycolysis; hypoxia-inducible factor-1α; psoriasis.

Figure 1. Expressions of HIF-1α, CD147, and GLUT1 protein in normal human skin (NHS) and psoriasis (immunohistochemistry, ×200, ×400, respectively)

A, D, G, J: HIF-1α is expressed in nucleus; B, E, H, K: CD147 is expressed in membranes; C, F, I, L: GLUT1 is expressed in membranes. Positive signals appear brown; blue counter staining with hematoxylin.

Figure 2. CD147 and GLUT1 were mediated by HIF-1α under hypoxia

A: HaCaT cells were treated with 250 μmol/L CoCl₂ for 12 h or ddH₂O without previous siRNA transfection and transcripts of HIF-1α, CD147, and GLUT1 were quantified by real-time RT-PCR. B: HaCaT cells were treated with 250 μmol/L CoCl₂ for 12 h or ddH₂O with previous siRNA transfection [siRNA against human HIF-1α or scrambled siRNA (siNC) for 48 h], transcripts of HIF-1α, CD147, and GLUT1 were quantified by real-time RT-PCR. C: Western blotting was performed with indicated antibodies for HaCaT cells only treated with 250 μmol/L CoCl₂ or ddH₂O. D: Western blotting was performed with indicated antibodies for HaCaT cells transfected with siRNA in hypoxia induced by 250 μmol/L CoCl₂. *P<0.05.

Figure 3. Interaction between CD147 and GLUT1

A: Co-localization of CD147 and GLUT1 in psoriatic epidermis (immunofluorescence, ×200, ×400, respectively). B: HaCaT cells were transfected with siRNA against human CD147 and scrambled siRNA as negative control (siNC), or CD147 cDNA plasmid and empty vector (EV), after 48 h, then treated with 250 μmol/L CoCl₂ for 12 h. Protein expressions of GLUT1 and CD147 were analyzed by Western blotting. C: HaCaT cells were transfected with siRNA against human GLUT1 or GLUT1 cDNA plasmid (negative controls and hypoxia condition as described before). Western blotting analysis was performed. D: HaCaT cells were treated with CoCl₂ for 12 h, and cell lysates were performed co-IP. Lysates were immunoprecipitated with antibodies of GLUT1 and rabbit IgG, and subjected to antibodies of CD147 and GLUT1 for immunoblotting. Anti-GLUT1 isolated CD147 while rabbit IgG did not.

Figure 4. Relative level of ATP in hypoxic HaCaT cells and different kinds of skin epidermis

A: Cellular ATP level in hypoxic HaCaT cells induced by 250 μmol/L CoCl₂ for 12 h, or in negative control with ddH₂O. B: HaCaT cells were treated with 250 μmol/L CoCl₂ for 12 h with previous siRNA transfection [siRNA against human HIF-1α or scrambled siRNA (siNC) for 48 h]. Cellular ATP level was detected. C: Cellular ATP level in normal skin epidermis (N), psoriatic non-lesional epidermis (PN), and psoriatic lesional epidermis (PS). Values present as mean±SD of 3 independent experiments. *P<0.05.

Figure 5. Relative level of lactate in hypoxic HaCaT cells and different kinds of skin epidermis

A: Cellular lactate level in hypoxic HaCaT cells induced by 250 μmol/L CoCl₂ for 12 h, or in negative control with ddH₂O. B: HaCaT cells were treated with 250 μmol/L CoCl₂ for 12 h with previous siRNA transfection [siRNA against human HIF-1α or scrambled siRNA (siNC) for 48 h]. Cellular lactate level was detected. C: Cellular lactate level in normal skin epidermis (N), psoriatic non-lesional epidermis (PN), and psoriatic lesional epidermis (PS). Values present as the mean±SD of 3 independent experiments. *P<0.05.