Regulation of Ligand and Shear Stress-induced Insulin-like Growth Factor 1 (IGF1) Signaling by the Integrin Pathway.

Mechanical loading of the skeleton, as achieved during daily movement and exercise, preserves bone mass and stimulates bone formation, whereas skeletal unloading from prolonged immobilization leads to bone loss. A functional interplay between the insulin-like growth factor 1 receptor (IGF1R), a major player in skeletal development, and integrins, mechanosensors, is thought to regulate the anabolic response of osteogenic cells to mechanical load. The mechanistic basis for this cross-talk is unclear. Here we report that integrin signaling regulates activation of IGF1R and downstream targets in response to both IGF1 and a mechanical stimulus. In addition, integrins potentiate responsiveness of IGF1R to IGF1 and mechanical forces. We demonstrate that integrin-associated kinases, Rous sarcoma oncogene (SRC) and focal adhesion kinase (FAK), display distinct actions on IGF1 signaling; FAK regulates IGF1R activation and its downstream effectors, AKT and ERK, whereas SRC controls signaling downstream of IGF1R. These findings linked to our observation that IGF1 assembles the formation of a heterocomplex between IGF1R and integrin ß3 subunit indicate that the regulation of IGF1 signaling by integrins proceeds by direct receptor-receptor interaction as a possible means to translate biomechanical forces into osteoanabolic signals.

Insulin-like growth factor-1 receptor in mature osteoblasts is required for periosteal bone formation induced by reloading.

Skeletal loading and unloading has a pronounced impact on bone remodeling, a process also regulated by insulin-like growth factor 1 (IGF-1) signaling. Skeletal unloading leads to resistance to the anabolic effect of IGF-1, while reloading after unloading restores responsiveness to IGF-1. However, a direct study of the importance of IGF-1 signaling in the skeletal response to mechanical loading remains to be tested. In this study, we assessed the skeletal response of osteoblast-specific Igf-1 receptor deficient (Igf-1r-/- ) mice to unloading and reloading. The mice were hindlimb unloaded for 14 days and then reloaded for 16 days. Igf-1r-/- mice displayed smaller cortical bone and diminished periosteal and endosteal bone formation at baseline. Periosteal and endosteal bone formation decreased with unloading in Igf-1r+/+ mice. However, the recovery of periosteal bone formation with reloading was completely inhibited in Igf-1r-/- mice, although reloading-induced endosteal bone formation was not hampered. These changes in bone formation resulted in the abolishment of the expected increase in total cross-sectional area with reloading in Igf-1r-/- mice compared to the control mice. These results suggest that the Igf-1r in mature osteoblasts has a critical role in periosteal bone formation in the skeletal response to mechanical loading.

Disruption of the hedgehog signaling pathway contributes to the hair follicle cycling deficiency in Vdr knockout mice.

Mice null for the Vitamin D receptor (VdrKO) have a disrupted first hair follicle cycle and aborted subsequent hair follicle cycling. We examined the expression of different markers and mediators of hair follicle cycling in the hair follicle of the VdrKO mouse during days 13-22 when the hair follicle normally initiates and completes the first catagen. We compared the expression of those genes in mice with a nonsense mutation in hairless (Rhino), which have a similar alopecia phenotype, and to Cyp27b1 null mice which are deficient in the production of 1,25(OH)2D3, the Vdr ligand, but display normal hair follicle cycling. Our results demonstrate the down regulation of hair follicle markers and the alteration of expression of hedgehog (Hh), Wnt, Fgf, and Tgfbeta pathways in VdrKO and Rhino mice, but not in Cyp27b1KO mice. Treatment of VdrKO mice with an agonist to the Hh pathway partially restored hair follicle cycling, suggesting a role of this pathway in the regulation of hair follicle cycling by VDR. These results suggest that Vdr regulates directly or indirectly the expression of genes required for hair follicle cycling, including Hh signaling, independent of 1,25(OH)2D3.

Injury enhances TLR2 function and antimicrobial peptide expression through a vitamin D-dependent mechanism.

An essential element of the innate immune response to injury is the capacity to recognize microbial invasion and stimulate production of antimicrobial peptides. We investigated how this process is controlled in the epidermis. Keratinocytes surrounding a wound increased expression of the genes coding for the microbial pattern recognition receptors CD14 and TLR2, complementing an increase in cathelicidin antimicrobial peptide expression. These genes were induced by 1,25(OH)2 vitamin D3 (1,25D3; its active form), suggesting a role for vitamin D3 in this process. How 1,25D3 could participate in the injury response was explained by findings that the levels of CYP27B1, which converts 25OH vitamin D3 (25D3) to active 1,25D3, were increased in wounds and induced in keratinocytes in response to TGF-beta1. Blocking the vitamin D receptor, inhibiting CYP27B1, or limiting 25D3 availability prevented TGF-beta1 from inducing cathelicidin, CD14, or TLR2 in human keratinocytes, while CYP27B1-deficient mice failed to increase CD14 expression following wounding. The functional consequence of these observations was confirmed by demonstrating that 1,25D3 enabled keratinocytes to recognize microbial components through TLR2 and respond by cathelicidin production. Thus, we demonstrate what we believe to be a previously unexpected role for vitamin D3 in innate immunity, enabling keratinocytes to recognize and respond to microbes and to protect wounds against infection.

IGF-I receptor is required for the anabolic actions of parathyroid hormone on bone.

UNLABELLED: We showed that the IGF-IR-null mutation in mature osteoblasts leads to less bone and decreased periosteal bone formation and impaired the stimulatory effects of PTH on osteoprogenitor cell proliferation and differentiation. INTRODUCTION: This study was carried out to examine the role of IGF-I signaling in mediating the actions of PTH on bone. MATERIALS AND METHODS: Three-month-old mice with an osteoblast-specific IGF-I receptor null mutation (IGF-IR OBKO) and their normal littermates were treated with vehicle or PTH (80 microg/kg body weight/d for 2 wk). Structural measurements of the proximal and midshaft of the tibia were made by microCT. Trabecular and cortical bone formation was measured by bone histomorphometry. Bone marrow stromal cells (BMSCs) were obtained to assess the effects of PTH on osteoprogenitor number and differentiation. RESULTS: The fat-free weight of bone normalized to body weight (FFW/BW), bone volume (BV/TV), and cortical thickness (C.Th) in both proximal tibia and shaft were all less in the IGF-IR OBKO mice compared with controls. PTH decreased FFW/BW of the proximal tibia more substantially in controls than in IGF-IR OBKO mice. The increase in C.Th after PTH in the proximal tibia was comparable in both control and IGF-IR OBKO mice. Although trabecular and periosteal bone formation was markedly lower in the IGF-IR OBKO mice than in the control mice, endosteal bone formation was comparable in control and IGF-IR OBKO mice. PTH stimulated endosteal bone formation only in the control animals. Compared with BMSCs from control mice, BMSCs from IGF-IR OBKO mice showed equal alkaline phosphatase (ALP)(+) colonies on day 14, but fewer mineralized nodules on day 28. Administration of PTH increased the number of ALP(+) colonies and mineralized nodules on days 14 and 28 in BMSCs from control mice, but not in BMSCs from IGF-IR OBKO mice. CONCLUSIONS: Our results indicate that the IGF-IR null mutation in mature osteoblasts leads to less bone and decreased bone formation, in part because of the requirement for the IGF-IR in mature osteoblasts to enable PTH to stimulate osteoprogenitor cell proliferation and differentiation.

Evaluation of fetal bone structure and mineralization in IGF-I deficient mice using synchrotron radiation microtomography and Fourier transform infrared spectroscopy.

The role of insulin like growth factor-I (IGF-I) during pre-natal development has not been evaluated in detail. However, the high degree of growth retardation and peri-natal mortality in IGF-I deficient mouse models indicates that it plays a critical role during this time. Techniques to assess the structure and quality of bone in small animal fetuses could be beneficial in better understanding its role in bone metabolism and skeletal development. Synchrotron microtomography (SR-microCT) and Fourier transform infrared spectroscopy (FTIR) may provide methods to visualize and quantify differences in the structure and mineral density of bone in small animal fetuses. Tibia and spine from IGF-I deficient and wildtype fetal mice (18th day gestation) were imaged using SR-microCT. Three-dimensional structural indices and the degree of mineralization were determined for each sample. Mineralization was also assessed using FTIR and von Kossa staining. Bone volume was systematically lower in IGF-I -/- animals (tibia: -15%, p<0.05) while both sites were found to have a more rod-like architecture (24%, p<0.05; 113%, p<0.01) and lower trabecular separation (-16%, p<0.05; -21%, p<0.05). These structural results were mostly consistent with those seen in adult models of IGF-I deficiency. The degree of mineralization as measured by SR-microCT was higher in the IGF-I tibial metaphysis (11.7%, p<0.0001), while FTIR of the whole bone showed mineralization to be lower in the knockout group (-11%, p<0.05). Interestingly, von Kossa staining revealed no mineral content in the IGF-I -/- spinal ossification center while SR-microCT clearly indicated the presence of highly attenuating components, if somewhat lower in IGF-I -/- animals (-2.2%, p<0.05). This indicates that IGF-I deficiency is linked to subtle differences in the mineral environment and mineralization progression. The advantages unique to SR-microCT allow for 3D visualization and quantification of pre-natal bone microstructure and mineral density in mice which was not previously possible.

Insulin-like growth factor I stimulates recovery of bone lost after a period of skeletal unloading.

IGF-I stimulates osteoblast proliferation, bone formation, and increases bone volume in normal weight-bearing animals. During skeletal unloading or loss of weight bearing, bone becomes unresponsive to the anabolic effects of insulin-like growth factor I (IGF-I). To determine whether skeletal reloading after a period of unloading increases bone responsiveness to IGF-I, we examined bone structure and formation in response to IGF-I under different loading conditions. Twelve-week-old rats were divided into six groups: loaded (4 wk), unloaded (4 wk), and unloaded/reloaded (2/2 wk), and treated with IGF-I (2.5 mg x kg(-1) x day(-1)) or vehicle during the final 2 wk. Cortical bone formation rate (BFR), cancellous bone volume and architecture in the secondary spongiosa (tibia and vertebrae), and total volume and calcified volume in the primary spongiosa (tibia) were assessed. Periosteal BFR decreased during unloading, remained low during reloading in the vehicle-treated group, but was dramatically increased in IGF-I-treated animals. Cancellous bone volume decreased with unloading and increased with reloading, but the effect was exaggerated in the tibia of IGF-I-treated animals. Total and calcified volumes in the primary spongiosa decreased during unloading in the vehicle-treated animals. IGF-I treatment prevented the loss in volume. These data show that reloading after a period of skeletal unloading increases bone responsiveness to IGF-I, and they suggest that IGF-I may be of therapeutic use in patients who have lost bone as a consequence of prolonged skeletal disuse.

Role of IGF-I signaling in regulating osteoclastogenesis.

UNLABELLED: We showed that IGF-I deficiency impaired osteoclastogenesis directly and/or indirectly by altering the interaction between stromal/osteoblastic cells and osteoclast precursors, reducing RANKL and M-CSF production. These changes lead to impaired bone resorption, resulting in high BV/TV in IGF-I null mice. INTRODUCTION: Although IGF-I has been clearly identified as an important growth factor in regulating osteoblast function, information regarding its role in osteoclastogenesis is limited. Our study was designed to analyze the role of IGF-I in modulating osteoclastogenesis using IGF-I knockout mice (IGF-I(-/-)). MATERIALS AND METHODS: Trabecular bone volume (BV/TV), osteoclast number, and morphology of IGF-I(-/-) or wildtype mice (IGF-I(+/+)) were evaluated in vivo by histological analysis. Osteoclast precursors from these mice were cultured in the presence of RANKL and macrophage-colony stimulating factor (M-CSF) or co-cultured with stromal/osteoblastic cells from either genotype. Osteoclast formation was assessed by measuring the number of multinucleated TRACP+ cells and pit formation. The mRNA levels of osteoclast regulation markers were determined by quantitative RT-PCR. RESULTS: In vivo, IGF-I(-/-) mice have higher BV/TV and fewer (76% of IGF-I(+/+)) and smaller osteoclasts with fewer nuclei. In vitro, in the presence of RANKL and M-CSF, osteoclast number (55% of IGF-I(+/+)) and resorptive area (30% of IGF-I(+/+)) in osteoclast precursor cultures from IGF-I(-/-) mice were significantly fewer and smaller than that from the IGF-I(+/+) mice. IGF-I (10 ng/ml) increased the size, number (2.6-fold), and function (resorptive area, 2.7-fold) of osteoclasts in cultures from IGF-I(+/+) mice, with weaker stimulation in cultures from IGF-I(-/-) mice. In co-cultures of IGF-I(-/-) osteoblasts with IGF-I(+/+) osteoclast precursors, or IGF-I(+/+) osteoblasts with IGF-I(-/-) osteoclast precursors, the number of osteoclasts formed was only 11% and 48%, respectively, of that from co-cultures of IGF-I(+/+) osteoblasts and IGF-I(+/+) osteoclast precursors. In the long bones from IGF-I(-/-) mice, mRNA levels of RANKL, RANK, M-CSF, and c-fms were 55%, 33%, 60%, and 35% of that from IGF-I(+/+) mice, respectively. CONCLUSIONS: Our results indicate that IGF-I regulates osteoclastogenesis by promoting their differentiation. IGF-I is required for maintaining the normal interaction between the osteoblast and osteoclast to support osteoclastogenesis through its regulation of RANKL and RANK expression.

Insulin-like growth factor-I is essential for embryonic bone development.

Although IGF-I has been identified as an important growth factor for the skeleton, the role of IGF-I on embryonic bone development remains unknown. Here we show that, in IGF-I-deficient (IGF-I(-/-)) mice, skeletal malformations, including short-limbed dwarfism, were evident at days post coitus (dpc) 14.5 to 18.5, accompanied by delays of mineralization in the spinal column, sternum, and fore paws. Reduced chondrocyte proliferation and increased chondrocyte apoptosis were identified in both the spinal ossification center and the growth plate of long bones. Abnormal chondrocyte differentiation and delayed initiation of mineralization was characterized by small size and fewer numbers of type X collagen expressing hypertrophic chondrocytes and lower osteocalcin expression. The Indian hedgehog-PTHrP feedback loop was altered; expression of Indian hedgehog was reduced in IGF-I(-/-) mice in long bones and in the spine, whereas expression of PTHrP was increased. Our results indicate that IGF-I plays an important role in skeletal development by promoting chondrocyte proliferation and maturation while inhibiting apoptosis to form bones of appropriate size and strength.

Gender differences in the response of CD-1 mouse bone to parathyroid hormone: potential role of IGF-I.

Parathyroid hormone (PTH) exerts both catabolic and anabolic actions on bone. Studies on the skeletal effects of PTH have seldom considered the effects of gender. Our study was designed to determine whether the response of mouse bone to PTH differed according to sex. As a first step, we analyzed gender differences with respect to bone mass and structural properties of 4 month old PTH treated (80 microg/kg per day for 2 weeks) male and female CD-1 mice. PTH significantly increased fat free weight/body weight, periosteal bone formation rate, mineral apposition rate, and endosteal single labeling surface, while significantly decreasing medullary area in male mice compared with vehicle treated controls, but induced no significant changes in female mice. We then analyzed the gender differences in bone marrow stromal cells (BMSC) isolated from 4 month old male and female CD-1 mice following treatment with PTH (80 microg/kg per day for 2 weeks). PTH significantly increased the osteogenic colony number and the alkaline phosphatase (ALP) activity (ALP/cell) by day 14 in cultures of BMSCs from male and female mice. PTH also increased the mRNA level of receptor activator of nuclear factor kappaB ligand in the bone tissue (marrow removed) of both females and males. However, PTH increased the mRNA levels of IGF-I and IGF-IR only in the bones of male mice. Our results indicate that on balance a 2-weeks course of PTH is anabolic on cortical bone in this mouse strain. These effects are more evident in the male mouse. These differences between male and female mice may reflect the greater response to PTH of IGF-I and IGF-IR gene expression in males enhancing the anabolic effect on cortical bone.

IGF-I Signaling in Osterix-Expressing Cells Regulates Secondary Ossification Center Formation, Growth Plate Maturation, and Metaphyseal Formation During Postnatal Bone Development.

To investigate the role of IGF-I signaling in osterix (OSX)-expressing cells in the skeleton, we generated IGF-I receptor (IGF-IR) knockout mice ((OSX)IGF-IRKO) (floxed-IGF-IR mice × OSX promoter-driven GFP-labeled cre-recombinase [(OSX)GFPcre]), and monitored postnatal bone development. At day 2 after birth (P2), (OSX)GFP-cre was highly expressed in the osteoblasts in the bone surface of the metaphysis and in the prehypertrophic chondrocytes (PHCs) and inner layer of perichondral cells (IPCs). From P7, (OSX)GFP-cre was highly expressed in PHCs, IPCs, cartilage canals (CCs), and osteoblasts (OBs) in the epiphyseal secondary ossification center (SOC), but was only slightly expressed in the OBs in the metaphysis. Compared with the control mice, the IPC proliferation was decreased in the (OSX)IGF-IRKOs. In these mice, fewer IPCs invaded into the cartilage, resulting in delayed formation of the CC and SOC. Immunohistochemistry indicated a reduction of vessel number and lower expression of VEGF and ephrin B2 in the IPCs and SOC of (OSX)IGF-IRKOs. Quantitative real-time PCR revealed that the mRNA levels of the matrix degradation markers, MMP-9, 13 and 14, were decreased in the (OSX)IGF-IRKOs compared with the controls. The (OSX)IGF-IRKO also showed irregular morphology of the growth plate and less trabecular bone in the tibia and femur from P7 to 7 weeks, accompanied by decreased chondrocyte proliferation, altered chondrocyte differentiation, and decreased osteoblast differentiation. Our data indicate that during postnatal bone development, IGF-I signaling in OSX-expressing IPCs promotes IPC proliferation and cartilage matrix degradation and increases ephrin B2 production to stimulate vascular endothelial growth factor (VEGF) expression and vascularization. These processes are required for normal CC formation in the establishment of the SOC. Moreover, IGF-I signaling in the OSX-expressing PHC is required for growth plate maturation and osteoblast differentiation in the development of the metaphysis.

Gender-Specific Differences in the Skeletal Response to Continuous PTH in Mice Lacking the IGF1 Receptor in Mature Osteoblasts.

The primary goal of this study was to determine whether the IGF1R in mature osteoblasts and osteocytes was required for the catabolic actions of continuous parathyroid hormone (cPTH). Igf1r was deleted from male and female FVN/B mice by breeding with mice expressing cre recombinase under control of the osteocalcin promoter ((0CN) Igfr1(-/-) ). Littermates lacking the cre recombinase served as controls. PTH, 60 µg/kg/d, was administered continuously by Alzet minipumps for 4 weeks. Blood was obtained for indices of calcium metabolism. The femurs were examined by micro-computed tomography for structure, immunohistochemistry for IGF1R expression, histomorphometry for bone formation rates (BFR), mRNA levels by qPCR, and bone marrow stromal cell cultures (BMSC) for alkaline phosphatase activity (ALP(+) ), mineralization, and osteoblast-induced osteoclastogenesis. Whereas cPTH led to a reduction in trabecular bone volume/tissue volume (BV/TV) and cortical thickness in the control females, no change was found in the control males. Although trabecular BV/TV and cortical thickness were reduced in the (0CN) Igfr1(-/-) mice of both sexes, no further reduction after cPTH was found in the females, unlike the reduction in males. BFR was stimulated by cPTH in the controls but blocked by Igf1r deletion in the females. The (0CN) Igfr1(-/-) male mice showed a partial response. ALP(+) and mineralized colony formation were higher in BMSC from control males than from control females. These markers were increased by cPTH in both sexes, but BMSC from male (0CN) Igfr1(-/-) also were increased by cPTH, unlike those from female (0CN) Igfr1(-/-) . cPTH stimulated receptor activator of NF-κB ligand (RANKL) and decreased osteoprotegerin and alkaline phosphatase expression more in control female bone than in control male bone. Deletion of Igf1r blocked these effects of cPTH in the female but not in the male. However, PTH stimulation of osteoblast-driven osteoclastogenesis was blocked by deleting Igfr1 in both sexes. We conclude that cPTH is catabolic in female but not male mice. Moreover, IGF1 signaling plays a greater role in the skeletal actions of cPTH in the female mouse than in the male mouse, which may underlie the sex differences in the response to cPTH.

Skeletal unloading induces resistance to insulin-like growth factor-I (IGF-I) by inhibiting activation of the IGF-I signaling pathways.

UNLABELLED: We showed that unloading markedly diminished the effects of IGF-I to activate its signaling pathways, and the disintegrin echistatin showed a similar block in osteoprogenitor cells. Furthermore, unloading decreased alphaVbeta3 integrin expression. These results show that skeletal unloading induces resistance to IGF-I by inhibiting activation of the IGF-I signaling pathways at least in part through downregulation of integrin signaling. INTRODUCTION: We have previously reported that skeletal unloading induces resistance to insulin-like growth factor-I (IGF-I) with respect to bone formation. However, the underlying mechanism remains unclear. The aim of this study was to clarify how skeletal unloading induces resistance to the effects of IGF-I administration in vivo and in vitro with respect to bone formation. MATERIALS AND METHODS: We first determined the response of bone to IGF-I administration in vivo during skeletal unloading. We then evaluated the response of osteoprogenitor cells isolated from unloaded bones to IGF-I treatment in vitro with respect to activation of the IGF-I signaling pathways. Finally we examined the potential role of integrins in mediating the responsiveness of osteoprogenitor cells to IGF-I. RESULTS: IGF-I administration in vivo significantly increased proliferation of osteoblasts. Unloading markedly decreased proliferation and blocked the ability of IGF-I to increase proliferation. On a cellular level, IGF-I treatment in vitro stimulated the activation of its receptor, Ras, ERK1/2 (p44/42 MAPK), and Akt in cultured osteoprogenitor cells from normally loaded bones, but these effects were markedly diminished in cells from unloaded bones. These results were not caused by altered phosphatase activity or changes in receptor binding to IGF-I. Inhibition of the Ras/MAPK pathway was more impacted by unloading than that of Akt. The disintegrin echistatin (an antagonist of the alphaVbeta3 integrin) blocked the ability of IGF-I to stimulate its receptor phosphorylation and osteoblast proliferation, similar to that seen in cells from unloaded bone. Furthermore, unloading significantly decreased the mRNA levels both of alphaV and beta3 integrin subunits in osteoprogenitor cells. CONCLUSION: These results indicate that skeletal unloading induces resistance to IGF-I by inhibiting the activation of IGF-I signaling pathways, at least in part, through downregulation of integrin signaling, resulting in decreased proliferation of osteoblasts and their precursors.

Insulin-like growth factor I is required for the anabolic actions of parathyroid hormone on mouse bone.

Parathyroid hormone (PTH) is a potent anabolic agent for bone, but the mechanism(s) by which it works remains imperfectly understood. Previous studies have indicated that PTH stimulates insulin-like growth factor (IGF) I production, but it remains uncertain whether IGF-I mediates some or all of the skeletal actions of PTH. To address this question, we examined the skeletal response to PTH in IGF-I-deficient (knockout [k/o]) mice. These mice and their normal littermates (NLMs) were given daily injections of PTH (80 microg/kg) or vehicle for 2 weeks after which their tibias were examined for fat-free weight (FFW), bone mineral content, bone structure, and bone formation rate (BFR), and their femurs were assessed for mRNA levels of osteoblast differentiation markers. In wild-type mice, PTH increased FFW, periosteal BFR, and cortical thickness (C.Th) of the proximal tibia while reducing trabecular bone volume (BV); these responses were not seen in the k/o mice. The k/o mice had normal mRNA levels of the PTH receptor and increased mRNA levels of the IGF-I receptor but markedly reduced basal mRNA levels of the osteoblast markers. Surprisingly, these mRNAs in the k/o bones increased several-fold more in response to PTH than the mRNAs in the bones from their wild-type littermates. These results indicate that IGF-I is required for the anabolic actions of PTH on bone formation, but the defect lies distal to the initial response of the osteoblast to PTH.

Lack of the vitamin D receptor is associated with reduced epidermal differentiation and hair follicle growth.

The active vitamin D metabolite, 1,25-dihydroxyvitamin D, acting through the vitamin D receptor, regulates the expression of genes in a variety of vitamin D-responsive tissues, including the epidermis. To investigate the role of the vitamin D receptor in mediating epidermal differentiation, we examined the histomorphology and expression of differentiation markers in the epidermis of vitamin D receptor knockout mice generated by gene targeting. The homozygous knockout mouse displayed a phenotype that closely resembles vitamin D-dependent rickets type II in humans, including the development of rickets and alopecia. Hair loss developed by 3 mo after birth and gradually led to nearly total hair loss by 8 mo. Histologic analysis of the skin of homozygous knockout mice revealed dilation of the hair follicles with the formation of dermal cysts starting at the age of 3 wk. These cysts increased in size and number with age. Epidermal differentiation markers, including involucrin, profilaggrin, and loricrin, detected by immunostaining and in situ hybridization, showed decreased expression levels in homozygous knockout mice from birth until 3 wk, preceding the morphologic changes observed in the hair follicles. Keratin 10 levels, however, were not reduced. At the ultrastructural level, homozygous knockout mice showed increased numbers of small dense granules in the granular layer with few or no surrounding keratin bundles and a loss of keratohyalin granules. Thus, both the interfollicular epidermis and the hair follicle appear to require the vitamin D receptor for normal differentiation. The temporal abnormalities between the two processes reflect the apparent lack of requirement for the vitamin D receptor during the anagen phase of the first (developmental) hair cycle, but with earlier effects on the terminal differentiation of the interfollicular epidermis.

Ephrin B2/EphB4 mediates the actions of IGF-I signaling in regulating endochondral bone formation.

Ephrin B2/EphB4 mediates interactions among osteoblasts (OBs), osteoclasts (OCLs), and chondrocytes to regulate their differentiation. We investigated the role of ephrin B2/EphB4 signaling in mediating the anabolic effects of insulin-like growth factor-I (IGF-I) and parathyroid hormone (PTH) on those cells and overall endochondral bone formation. Immunohistochemistry demonstrated that the expression of ephrin B2 in OBs, OCLs, and osteocytes, and the expression of EphB4 in OBs and osteocytes was dramatically decreased in global IGF-I knockout mice. Inactivation of EphB4 by EphB4 small, interfering RNA (siRNA) in cultured bone marrow stromal cells significantly decreased the mRNA levels of OB differentiation markers and abolished the stimulatory effects of IGF-I on these markers. Blocking the interaction of EphB4 and ephrin B2 in the OB-OCL cocultures with the EphB4 specific peptide TNYL-RAW or deletion of ephrin B2 in OCL prior to coculture led to fewer and smaller tartrate-resistant acid phosphatase (TRAP)-positive cells, decreased expression of OB differentiation markers, and blunted response to IGF-I for both OCL and OB differentiation. In the growth plate, both ephrin B2 and EphB4 are expressed in late stage proliferating and prehypertrophic chondrocytes, and their expression was decreased in mice lacking the IGF-I receptor specifically in chondrocytes. In vitro, blocking the interaction of EphB4 and ephrin B2 in chondrogenic ATDC5 cells with TNYL-RAW significantly decreased both basal and IGF1-induced expression of type II and type X collagen. In the cocultures of ATDC5 cells and spleen cells (osteoclast precursors), TNYL-RAW decreased the numbers of TRAP-positive cells and the expression of nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) and receptor activator of NF-κB (RANK), and blocked their stimulation by IGF-I. Our data indicate that IGF-I/IGF-IR signaling promotes OB, OCL, and chondrocyte differentiation via ephrin B2/EphB4 mediated cell-cell communication.

Protective role of vitamin D signaling in skin cancer formation.

Vitamin D sufficiency is associated with protection against malignancy in a number of tissues clinically, and a strong body of evidence from animal and cell culture studies supports this protective role. Cancers in the skin differ, however, in that higher serum levels of 25OHD are associated with increased basal cell carcinomas (BCC), the most common form of epidermal malignancy. This result may be interpreted as indicating the role of UVR (spectrum 280-320) in producing vitamin D in the skin as well as causing those DNA mutations and proliferative changes that lead to epidermal malignancies. Recent animal studies have shown that mice lacking the vitamin D receptor (VDR) are predisposed to developing skin tumors either from chemical carcinogens such as 7,12-dimethylbenzanthracene (DMBA) or chronic UVR exposure. Such studies suggest that vitamin D production and subsequent signaling through the VDR in the skin may have evolved in part as a protective mechanism against UVR induced epidermal cancer formation. In this manuscript we provide evidence indicating that vitamin D signaling protects the skin from cancer formation by controlling keratinocyte proliferation and differentiation, facilitating DNA repair, and suppressing activation of the hedgehog (Hh) pathway following UVR exposure. This article is part of a Special Issue entitled 'Vitamin D Workshop'.

Coactivator MED1 ablation in keratinocytes results in hair-cycling defects and epidermal alterations.

The transcriptional coactivator complex Mediator (MED) facilitates transcription of nuclear hormone receptors and other transcription factors. We have previously isolated the MED complex from primary keratinocytes (KCs) as the vitamin D receptor-interacting protein complex. We identified a role for MED in KC proliferation and differentiation in cultured KCs. Here, we investigated the in vivo role of MED by generating a conditional null mice model in which a critical subunit of the MED complex, MED1, is deleted from their KCs. The MED1 ablation resulted in aberrant hair differentiation and cycling, leading to hair loss. During the first hair follicle (HF) cycle, MED1 deletion resulted in a rapid regression of the HFs. Hair differentiation was reduced, and ß-catenin/vitamin D receptor (VDR)-regulated gene expression was markedly decreased. In the subsequent adult hair cycle, MED1 ablation activated the initiation of HF cycling. Shh signaling was increased, but terminal differentiation was not sufficient. Deletion of MED1 also caused hyperproliferation of interfollicular epidermal KCs, and increased the expression of epidermal differentiation markers. These results indicate that MED1 has a critical role in regulating hair/epidermal proliferation and differentiation.

Ablation of the calcium-sensing receptor in keratinocytes impairs epidermal differentiation and barrier function.

The calcium-sensing receptor (CaR) has an essential role in mediating Ca(2+)-induced keratinocyte differentiation in vitro. In this study, we generated keratinocyte-specific CaR knockout ((Epid)CaR(-/-)) mice to investigate the function of the CaR in epidermal development in vivo. (Epid)CaR(-/-) mice exhibited a delay in permeability barrier formation during embryonic development. Ion capture cytochemistry detected the loss of the epidermal Ca(2+) gradient in the (Epid)CaR(-/-) mice. The expression of terminal differentiation markers and key enzymes mediating epidermal sphingolipid transport and processing in the (Epid)CaR(-/-) epidermis was significantly reduced. The (Epid)CaR(-/-) epidermis displayed a marked decrease in the number of lamellar bodies (LBs) and LB secretion, thinner lipid-bound cornified envelopes, and a defective permeability barrier. Consistent with in vivo results, epidermal keratinocytes cultured from (Epid)CaR(-/-) mice demonstrated abnormal Ca(2+)(i) handling and diminished differentiation. The impairment in epidermal differentiation and permeability barrier in (Epid)CaR(-/-) mice maintained on a low calcium (0.02%) diet is more profound and persistent with age than in (Epid)CaR(-/-) mice maintained on a normal calcium (1.3%) diet. Deleting CaR perturbs the epidermal Ca(2+) gradient and impairs keratinocyte differentiation and permeability barrier homeostasis, indicating a key role for the CaR in normal epidermal development.

Overexpression of hedgehog signaling is associated with epidermal tumor formation in vitamin D receptor-null mice.

The vitamin D receptor (VDR) ligand, 1,25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), reduces proliferation and enhances differentiation, and thus has been investigated for a role in preventing or treating cancer. Mice deficient for the VDR display a hyperproliferative response in the hair follicle and epidermis and decreased epidermal differentiation. Unlike their wild-type littermates, when treated with 7,12 dimethylbenzanthracene (DMBA) or UVB, they develop skin tumors, including some characteristic of overexpression of the hedgehog (Hh) pathway. Both the epidermis and utricles of the VDR-null animals overexpress elements of the Hh pathway (sonic hedgehog (Shh) 2.02-fold, patched1 1.58-fold, smoothened 3.54-fold, glioma-associated oncogene homolog (Gli)1 1.17-fold, and Gli2 1.66-fold). This overexpression occurs at an age (11 weeks) at which epidermal hyperproliferation is most visible and is spatially controlled in the epidermis. DMBA- or UVB-induced tumors in the VDR-null mice also overexpress elements of this pathway. Moreover, 1,25(OH)(2)D(3) downregulates the expression of some members of the Hh pathway in an epidermal explants culture system, suggesting a direct regulation by 1,25(OH)(2)D(3). Our results suggest that increased expression of Shh in the keratinocytes of the VDR-null animal activates the Hh pathway, predisposing the skin to the development of both malignant and benign epidermal neoplasms.