Mamoru Kiyoki

1,24(R)-Dihydroxyvitamin D3, a Novel Active Form of Vitamin D3 with High Activity for Inducing Epidermal Differentiation but Decreased Hypercalcemic Activity

1α,25‐Dihydroxyvitamin D3 (1,25(OH)2D3) is known to be a hormonally active form of vitamin D3 in the regulation of intracellular and extracellular calcium levels and of differentiation of myeloid cells and epidermal keratinocytes. We found that 1α,24(R)‐dihydroxyvitamin D3 (1,24(OH)2D3), a novel synthetic derivative of vitamin D3, is also active in regulating the differentiation of epidermal keratinocytes. 1,24(OH)2D3 had the same affinity as 1,25(OH)2D3 for a receptor isolated from the epidermis of newborn mice. The incubation of mouse epidermal keratinocytes with 1,24(OH)2D3 induced their differentiation in a time‐ and dose‐dependent manner, as determined by the formation of a cornified envelope and an increase in the activity of transglutaminase. 1,24(OH)2D3 inhibited DNA synthesis of epidermal keratinocytes and also increased their cytosolic calcium level. These effects of 1,24(OH)2D3 were similar to, or rather more than, those of physiologically active 1,25(OH)2D3. However, 1,24(OH)2D3 was found to cause less hypercalcemia than 1,25(OH)2D3 when administrated intravenously to rats, suggesting its possible therapeutic value in psoriasis.

Effect of 1,24R-dihydroxyvitamin D3 on the Growth of Human Keratinocytes

The effect of 1,24R‐dihydroxyvitamin D3 (1,24R(OH)2D3), a synthetic analogue of a biologically active form of vitamin D3 (1,25‐dihydroxyvitamin D3, 1,25(OH)2D3), on the growth of human keratinocytes cultured in serum‐free medium was investigated. The growth of cultured normal human keratinocytes was inhibited by 65% by 10−8M 1,24R(OH)2D3 and by 90% by 10−7M 1,24(OH)2D3. It inhibited cell growth almost completely at 10−6M. The DNA synthesis of keratinocytes was also inhibited with 1,24R(OH)2D3 by 27% at 10−8M, 59% at 10−7M, and 92% at 10−6M. The inhibition of cell growth and DNA synthesis were more remarkable by 1,24R(OH)2D3 than by 1,25(OH)2D3. 1,24R(OH)2D3 also inhibited the growth of keratinocytes derived from patients with psoriasis vulgaris; the growth inhibitory effect was again more remarkable with 1,24R(OH)2D3 than with 1,25(OH)2D3. The viability and protein synthesis of keratinocytes were not affected by 1,24R(OH)2D3, suggesting that the growth inhibitory effect is due to its biological activity, not to cytotoxicity. The binding of [3H]‐labeled 1,25(OH)2D3 to its receptor in the cytosolic fraction of cultured keratinocytes was competitively substituted by unlabeled 1,24R(OH)2D3 as well as 1,25(OH)2D3, suggesting that 1,24R(OH)2D3 binds to the 1,25(OH)2D3 receptor. It was found that the affinity of 1,24R(OH)2D3 for the receptor was slightly higher than that of 1,25(OH)2D3. These results demonstrate that 1,24R(OH)2D3 functions as a potent growth inhibitor in vitro in human keratinocytes from both normal and psoriatic epidermis, and it possesses a higher affinity for the 1,25(OH)2D3 receptor in cultured human keratinocytes. The difference in affinity of 1,24R(OH)2D3 for the 1,25(OH)2D3 receptor correlates with its greater inhibition of keratinocyte growth than 1,25(OH)2D3. 1,24R(OH)2D3 may be useful in the treatment of psoriasis.

1α,24R-Dihydroxyvitamin D3 Has an Ability Comparable to That of 1α,25-Dihydroxyvitamin D3 to Induce Keratinocyte Differentiation

Using cultured normal human keratinocytes, we compared the activities of 1α,24R‐dihydroxyvitamin D3 (1,24R(OH)2D3), a synthetic derivative of vitamin D3, and 1α,25‐dihydroxyvitamin D3 (1,25(OH)2D3), a biologically active form, in inducing cell differentiation. Treatment with 10−6 M of 1,24R(OH)2D3 and 1,25(OH)2D3 increased the number of involucrin positive cells (differentiated cells) from 6.4% to 24.1% and 25.1%, respectively. These results indicate that 1,24R(OH)2D3 has an ability comparable to that of 1,25(OH)2D3 to induce cell differentiation in human keratinocytes. The clinical effectiveness of 1,24R(OH)2D3 for the treatment of psoriasis may be, in part, related to its direct effect on hyperproliferative keratinocytes.

Tacalcitol, an active vitamin D3, induces nerve growth factor production in human epidermal keratinocytes.

The human epidermal keratinocyte cell line K-TL-1, developed from a benign epidermal tumor, was cultured in the presence of the synthetic vitamin D3 analogue tacalcitol [1α,24(R)-dihydroxyvitamin D3] to assess the effects on the production of nerve growth factor (NGF). Confluent K-TL-1 cells were cultured with 10–8M of tacalcitol. Supernatants and cell homogenates were collected and NGF concentrations were determined by enzyme-linked immunosorbent assay. The concentration of NGF in the supernatants of cultures treated with tacalcitol peaked within 24 h after the start of tacalcitol treatment and remained stable for 96 h. This NGF induction caused by tacalcitol was dose-dependent, showing an ED50 between 10–10 and 10–9M. Induction of NGF mRNA expression by tacalcitol was also observed by RT-PCR, indicating that tacalcitol induced NGF expression through transcriptional activation. These results suggest that active vitamin D3 could treat peripheral neuropathy by inducing NGF production in the skin.

Sandwich immunoassay specific for the N-terminal sequence of osteocalcin.

A monoclonal antibody (10B) against the N-terminal sequence of human osteocalcin was selected to characterize its epitope and species specificity. The cross-reactivity of 10B with human and rat osteocalcin demonstrated that the reactivities of 10B with both human and rat osteocalcins were very similar. The pin technology method was used to determine the epitope and clearly demonstrated that the epitope recognized by 10B was localized to residues 12-16, the sequence of which is identical in rat and human osteocalcin molecules. This monoclonal antibody was found to be useful for designing region-specific sandwich immunoassay systems for human and rat N-terminal osteocalcin (N-OC) molecules using rabbit anti-hN20 and anti-rN20 polyclonal antibodies, respectively. The osteocalcin levels in serum determined by this N-OC method were stable during prolonged storage at 25 degrees C and the method could be usefully applied in the development of immunoassay systems for many osteocalcin molecules from many other species.

Tacalcitol (1,24(OH)2D3, TV-02) inhibits phorbol ester-induced epidermal proliferation and cutaneous inflammation, and induces epidermal differentiation in mice

In this study, we examined the cutaneous effects of tacalcitol [1, 24(R)(OH)2D3] on epidermal proliferation, differentiation, and skin inflammation in vivo using hairless mice. Tacalcitol was shown to inhibit epidermal proliferation using TPA-induced ornithine decarboxylase activity and DNA synthesis as indices, and the induction of epidermal differentiation using type I transglutaminase activity as an index. Tacalcitol also displayed an antiinflammatory effect on TPA-induced inflammatory changes histopathologically. These results confirm the clinical efficacy of tacalcitol in psoriasis, and suggest that it may be efficacious in the treatment of other inflammatory skin diseases.

Species specificity of anticoagulant activity of activated human protein C. Involvement of factor V as well as protein S

Activated protein C (APC) possesses species specificity in its anticoagulant activity. Human APC exerts only weak activity in rat plasma compared with that in human plasma. The present study was undertaken to estimate the difference in interaction of human and rat factors with human APC and to assess the cause of the species specificity. Human or rat protein S (PS), factor V, or factor VIII was used to supplement human plasma depleted of each respective factor, and the anticoagulant activity of human APC was measured in term of the elongation of activated partial thromboplastin time (APTT). The activity of human APC in rat PS- or factor V-supplemented plasma was weaker than that in the human PS- or factor V-supplemented plasma. Furthermore, using purified human and rat factor V, human APC showed weaker inactivation of rat factor V than human factor V. Equal anticoagulant activity was observed in human or rat factor VIII-supplemented plasma. And there was a little difference in the interaction of APC with its inhibitors in human or rat plasma during a few minutes of incubation as judged by measurement of residual activity by an enzyme capture assay. From these results factor V as well as PS seems to play a major role in the species specificity of APC.

In vivo microautoradiography of [3H]1,24(OH)2D3 (tacalcitol) following topical application to normal rats and in vitro metabolism in human keratinocytes

This study was conducted to investigate the mechanism of topical absorption of [3H]1,24(OH)2D3 (1,24-dihydroxyvitamin D3; tacalcitol) by applying an ointment containing 4 μg/g [3H]1,24(OH)2D3 to the skin of rats using an occlusion method. Microautoradiography of the skin at the application site 1 h after topical treatment showed a high concentration of radiolabel in the stratum corneum, the epidermis and around the hair follicles. Radiolabel was also seen in the epidermis and hair follicle areas 8 h and 24 h after application. The radiolabel was distributed to a minor extent to the subcutaneous fat layer. Microautoradiography showed two routes of purcutaneous absorption of 1,24(OH)2D3: through the stratum corneum and epidermis into the microvessels, and through hair follicle areas into the bloodstream. After topical application of an ointment containing 4 μg/g or 40 μg/g [3H]1,24(OH)2D3 to the shaved neck skin of rats, the absorption rate, estimated by excretion in the urine and faeces, was about 30% of the total applied radioactivity. The main excretion route after topical application was in the faeces. Furthermore, 1,24(OH)2D3 added to human adult keratinocytes was not metabolized into other compounds, and only the unchanged compound was detected. These findings strongly suggest that 1,24(OH)2D3 distributed into the epidermis acts on epidermal keratinocytes. Topical application of 1,24(OH)2D3 appears to be a possible approach to the treatment of psoriasis and other skin diseases through its action on the 1,25(OH)2D3 receptor, which reportedly plays a very important role in the regulation of proliferation and differentiation of keratinocytes.

Comparison of hemorrhagic effect of heparin and human activated protein C with use of Thrombostat 4000.

The importance of bleeding as a complication of anticoagulant therapy is clearly recognized. We previously reported that amelioration of hemorrhage associated with disseminated intravascular coagulation by the human activated protein C (APC) was greater than that by heparin. In this study, we compared the bleeding complication of intravenously administered APC and heparin in rabbits, and also estimated primary hemostasis. When both anticoagulants were intravenously infused, the bleeding time from a punctured ear vein was prolonged dose-dependently. However, at doses which prolonged the activated partial thromboplastin time nearly equally, the prolongation of bleeding was greater in heparin-administered rabbits. Blood withdrawn from heparin-administered animals showed increases in in vitro bleeding parameters which correlated with the in vivo bleeding time. However, only small changes were observed in the blood withdrawn from APC-administered animals. Both drugs induced either no change or only a slight decrease in the platelet count, hematocrit and fibrinogen content. These observations suggest that APC may be a more useful anticoagulant than heparin since it causes less bleeding tendency.