Hair growth defects in Insig-deficient mice caused by cholesterol precursor accumulation and reversed by simvastatin

Abstract

Insig-1 and Insig-2, two closely related proteins, are essential for feedback inhibition of cholesterol biosynthesis. Here, we characterized a line of epidermal-specific, Insig-double knockout (Epi-Insig-DKO) mice lacking both Insigs in epidermis. At birth, Epi-Insig-DKO mice were indistinguishable from control littermates, but thereafter they failed to thrive and died before 6 weeks of age. By 14 days of age, 100% of Epi-Insig-DKO mice exhibited defects in hair growth along with other skin abnormalities, including hyperkeratosis. Hair follicles in Epi-Insig-DKO mice developed normally through postnatal day 7, but they failed to progress to later stages and thus exhibited defects in postnatal hair cycling. Insig deficiency caused a marked buildup of cholesterol precursors in skin associated with a marked increase in 3-hydroxy-3-methylglutaryl coenzyme A reductase protein. Topical treatment of Epi-Insig-DKO mice with simvastatin, an inhibitor of reductase, reduced sterol precursors in skin and corrected the hair and skin defects. We conclude that Insig deficiency in skin causes accumulation of cholesterol precursors, and this impairs normal hair development. These findings have implications for several human genetic diseases in which mutations in cholesterol biosynthetic enzymes lead to accumulation of sterol precursors and multiple cutaneous abnormalities.

Figure 1. Generation of mice lacking Insigs in the epidermis

(a) Schematic of epidermal-specific deletion of Insig-1. Mice carrying floxed Insig-1 and null Insig-2 alleles (Insig-1^f/f;Insig-2^−/−) were bred to Keratin14-Cre (K14-Cre) transgenic mice in which Cre recombinase driven by the human Keratin14 promoter was expressed in the oral epithelium, epidermis, and hair follicles. This Cre-mediated deletion of Insig-1, in combination with the germ-line deletion of Insig-2, generated mice that lacked both Insig-1 and Insig-2 in these tissues. Because the epidermis was the major tissue in which Insigs were ablated, we designated these mutant mice as epidermal-specific Insig-double knockout (Epi-Insig-DKO) mice. (b) Total RNA was isolated from back skins and livers of postnatal day 14 (PND 14) littermate control (Insig-1^f/f;Insig-2^−/−) and Epi-Insig-DKO mice. Relative amount of Insig-1 mRNA was determined by quantitative real-time PCR using the comparative C_t method. Each bar represents the mean ± SEM of data from five mice. The C_t (cycle threshold) numbers are shown inside the bars. (c) Body weights of littermate control and Epi-Insig-DKO mice were measured at various ages. Each bar represents the mean ± SEM of data from the indicated number of mice. Statistical analysis was performed with the two-tailed Student’s t-test.

Figure 2. Gross morphology of control and Epi-Insig-DKO mice

(a–c) Representative photographs of control and Epi-Insig-DKO littermates at postnatal day (PND) 14. Compared with control mice (a, left), all mutant mice (a, right) were smaller in size, exhibited thickening and flaking of the skin, and failed to grow body hair. Epi-Insig-DKO mice (c) also exhibited exophthalmos and short, kinked vibrissae compared with control mice (b). Scale bars=1.0 cm (a–c).

Figure 3. Histology of skin from control and Epi-Insig-DKO mice

Skin from the upper back of postnatal day (PND) 14 littermate control and Epi-Insig-DKO mice was fixed, sectioned parallel (a, b, e, f) or perpendicular (c, d) to the hair shaft, and stained with hematoxylin and eosin. Shown here are sections representative of five mice in each group. (a, b) Epi-Insig-DKO mice showed a loss of hypodermal adipocytes, misalignment, and ectasia of hair follicles (white arrowhead), and kinking of hair shafts (compare inset of b with the inset of a). (c, d) Epi-Insig-DKO mice exhibited epidermal hyperplasia (black double arrows). The dashed line represents the border between the epidermis and dermis. (e, f) The dermal papillae (white arrows) of Epi-Insig-DKO mice separated from the follicular epithelial cells of the hair bulb (hb), leading to its degeneration. Scale bars=0.1 mm (a, b) and 0.05 mm (c–f, insets of a and b).

Figure 4. Hair follicle morphogenesis and cycling in control and Epi-Insig-DKO mice

Skin from the upper back of postnatal days (PNDs) 4, 7, 14, 21, and 28 littermate mice was fixed, sectioned parallel to the hair shaft, and stained with hematoxylin and eosin. Shown here are sections representative of five mice in each group. (a, f) At PND 4, hair follicles were histologically identical between control (a) and Epi-Insig-DKO (f) mice. (b, g) At PND 7, some hair shafts in Epi-Insig-DKO mice (g) had become kinked (inset of g). (c, h) By PND 14, the hair follicles of Epi-Insig-DKO mice (h) had become completely misaligned. Furthermore, Epi-Insig-DKO mice also exhibited a loss of hypodermal adipocytes, hyperkeratosis, ectatic hair follicles (white arrowhead), kinking of the hair shafts (compare inset of h with the inset of c), and hair bulb degeneration. (d, e, i, j) At PND 21, when hair follicles of control mice (d) were in the telogen phase of the follicular cycle, those of Epi-Insig-DKO mice (i) were arrested and exhibited progressive ectasia of hair follicles (white arrowheads) with the accumulation of keratin within the follicle. This arrest of hair follicle cycling persisted in the mutant mice (j) at PND 28 with further exacerbation of hair follicle abnormalities; whereas, hair follicles of control mice (e) proceeded into the anagen phase normally. Scale bars=0.1 mm (a–j) and 0.05 mm (insets of b, c, g, h).

Figure 5. Staining of lipids and immunological visualization of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in skin from control and Epi-Insig-DKO mice

(a–d) Skin from the upper back of postnatal day (PND) 14 control and Epi-Insig-DKO mice was fixed, cryosectioned, and stained with oil red O. Shown here are representative sections from four mice per group. (a, c) Sebaceous glands (arrows) were enlarged in mutant (c) compared with control (a) mice. (b, d) Lipids accumulated in the epidermal compartment (arrow) of the hair bulbs in mutant (d) but not in control (b) mice. (e) Immunoblot analysis of HMG-CoA reductase in skin from the upper back of PND 14 control and Epi-Insig-DKO mice. Aliquots of whole-cell lysates (30 μg) were subjected to 8% SDS–PAGE and immunoblot analysis with a rabbit polyclonal antibody against mouse HMG-CoA reductase. Transferrin receptor was used as loading control. Shown are results from four control (lanes 1–4) and four Epi-Insig-DKO (lanes 5–8) mice. (f, g) Immunohistochemical analysis of HMG-CoA reductase in skin from upper back of PND 14 control (f) and Epi-Insig-DKO (g) mice. Shown are sections representative of five mice in each group. Sebaceous glands (arrows) of Epi-Insig-DKO mice (g) showed a massive increase in reductase protein compared with those of control mice (f). Scale bars=0.1 mm (a, c), 0.01 mm (b, d), and 0.02 mm (f, g).

Figure 6. Sterol content in the skin of control and Epi-Insig-DKO mice treated with or without simvastatin

(a) Pathway for cholesterol biosynthesis, showing site of inhibition by simvastatin. (b–g) Littermate control and mutant mice were treated daily with either 1 mg simvastatin or vehicle from postnatal day (PND) 2 to PND 13. At PND 14, skin from the upper back was harvested and subjected to gas chromatography–mass spectroscopy analysis to measure the levels of various sterols. Each bar represents the mean ± SEM of data from five mice. Statistical analysis was performed with the two-tailed Student’s t-test.

Figure 7. Gross morphology and histology of control and Epi-Insig-DKO mice treated with or without simvastatin

Littermate control and Epi-Insig-DKO mice were treated daily with either 1 mg simvastatin or vehicle from postnatal day (PND) 2 to PND 13. (a–d) At PND 14, animals were photographed and analyzed for hair growth by two independent observers before genotyping. Although none (0 out of 11) of the vehicle-treated Epi-Insig-DKO mice had body hair (c), 100% (16 out of 16) of the simvastatin-treated Epi-Insig-DKO mice exhibited normal hair (d). Hair growth in control mice was not affected by either vehicle (a) or simvastatin (b) treatment. (e–p) Mice were killed at PND 14, and skin from the upper back was fixed and either sectioned for hematoxylin and eosin staining (e–l) or cryosectioned for oil red O staining (m–p). Shown here are sections representative of three mice (m–p) or five mice (e–l) per group. Simvastatin treatment of mutant mice prevented the hair follicle disorganization and degeneration (h vs. g), the hair bulb degeneration and dermal papillae separation (l vs. k), and the accumulation of lipid droplets (black arrows) in the epidermal compartment of hair bulbs (p vs. o). Simvastatin treatment did not have any effect on these histological parameters in control mice (f, j, n vs. e, i, m). Scale bars=1.0 cm (a–d) and 0.1 mm (e–p).

Figure 8. Morphology and skin sterol content of control and Epi-Insig-DKO mice at 3 and 5.5 months after stopping simvastatin

(a, b) Five Epi-Insig-DKO mice were treated daily with 1 mg simvastatin from postnatal day (PND) 2 to PND 13. Thereafter, the mice were left untreated for 5.5 months. These photographs show the same Epi-Insig DKO mouse at 3.5 months (a) and 6 months (b) of age. The loss of hair and decrease in body weight that occurred in this mouse between 3.5 and 6 months of age was reproduced in all five mutant mice studied in this experiment. (c–h) The sterol content in the back skin from control and Epi-Insig-DKO mice at 6 months was measured by gas chromatography–mass spectroscopy. Each bar represents the mean ± SEM of data from five mice. Statistical analysis was performed with the two-tailed Student’s t-test.