Nicotinic acid modulates intracellular calcium concentration and disassembles the cytoskeleton

Nicotinic acid (NA), a member of the vitamin B family, is well known for its functions in the treatment and prevention of atherosclerosis due to decreasing plasma levels of low-density lipoprotein cholesterol. In recent years, the major side effect of NA, cutaneous flushing, has also attracted extensive attention. However, the effects of NA in other aspects of physiology or cell biology have remained elusive. The present study provided evidence that high concentrations of NA were able to first reduce and later elevate intracellular [Ca2+] in the NIH3T3 cell line. The reduction of the intracellular Ca2+ concentration was achieved within the initial 10 sec, and was preceded by a gradual elevation of intracellular [Ca2+]. Notably, marked accumulation of opaque materials in the perinuclear region was observed in NIH3T3 cells treated with 70 mM NA. Further analysis revealed that treatment with 70 mM NA for 1 h disassembled the microtubule and F-actin cytoskeleton systems and resulted in β-tubulin degradation in an ubiquitin-proteasome-dependent manner. These data indicated that high concentrations of NA disrupted cytoskeleton structures, which may have contributed to minus end (nucleus region) to plus end (cell membrane region)-directed transport processes and resulted in the deposition of material in the perinuclear region. Artificially increasing [Ca2+] adding CaCl2 to the culture media effected the disassembly of F-actin, while it had no apparent effect on microtubules. These results suggested that the disruption of the cytoskeleton systems was not entirely due to the NA-induced elevation of [Ca2+]. Finally, microinjection of NA into xenopus embryos blocked the transport of melanosomes to the peripheral cellular area. In conclusion, the present study indicated that NA disassembles F-actin and microtubule systems, thereby blocking cytoskeleton-dependent intracellular transport.


Introduction
The milestone results reported by Altschul et al (1) >50 years ago demonstrated that nicotinic acid (NA) has the capacity to decrease plasma lipids. As a result, this water soluble vitamin B family member has been widely used clinically for the treatment and prevention of atherosclerosis and other lipid-metabolic disorders (2,3). At present, NA is one of the most effective agents that offers protection against cardiovascular risk factors by increasing high density lipoprotein (HDL) levels, while simultaneously decreasing very low density lipoprotein (VLDL) and low density lipoprotein (LDL) levels (4). The major side effect of NA is cutaneous vasodilatation, also known as 'flush', which limits its clinical utility and applications (5). NA functions by downregulating intracellular cyclic adenosine monophosphate (cAMP), the major intracellular mediator of prolipolytic stimuli, and subsequently decreases cellular levels of free fatty acids (5).
Xenopus embryo manipulation and microinjection. In vitro embryo fertilization and culture were conducted as described previously (11). For each embryo, 70 ng of NA was injected at the 2 cell stage. The microinjection procedure was performed as described previously (12). The use of Xenopus embyos in the study was approved by the Ethics Comittee of the Children's Hospital of Chongqing Medical University (Chongqing, China). The NIH3T3 cells were further exposed to a wider span of NA concentration gradients and the Ca 2+ mobilization was assessed. At a 1 mM NA, the intracellular Ca 2+ levels decreased by 50% within 10 sec and no elevation was detected during the entire process ( Fig. 1A and B, green curve). In the 10 mM NA exposure group, intracellular free Ca 2+ reduced precipitously similarly to the observations at 1 mM NA; however, a transient sharp elevation-reduction n-turn like curve of Ca 2+ mobilization was observed ( Fig. 1A and B, light blue curve). Consistent with the 10 mM group, both 70 mM (Fig. 1B, purple curve) and 100 mM (Fig. 1B, Fig. 1A and C, pink curve), an endoplasmic reticulum (ER) Ca 2+ -ATPase pump inhibitor, which induces Ca 2+ release from the ER. Furthermore, the NA-induced decrease in primary intracellular [Ca 2+ ] was delayed by the addition of 2 mM of the cAMP analog 8Br-cAMP ( Fig. 1A and C, green curve). These data suggested that the reduction of cAMP levels by NA may be responsible for the primary transient ER decrease and Ca 2+ release by the endoplasmic reticulum (ER) contributed to the later observed Ca 2+ elevation.

NA regulates intracellular Ca
NA disassembles the cytoskeleton and deposits opaque materials at the perinuclear region. Besides intracellular Ca 2+ wave variation, the results revealed that an accumulation of unidentified opaque material at the perinuclear region, forming a ring-type structure, as well as at the nucleolus, was markedly evident in the NIH3T3 cells following incubation with 70 mM NA (Fig. 2B). However, in the phosphate-buffered saline (PBS)treated control group, the NIH3T3 cells exhibited a spread morphology and no perinuclear ring or dim nucleolus phenotypes ( Fig. 2A). The highly visible nucleolus in the NA-treated cells suggested the activation of synthetic processes; however, the manner in which the perinuclear opaque rings formed remains elusive. Cytoskeletal organization has a number of important roles in intracellular transport processes. The assembly-disassembly homeostasis of F-actin and microtubules are regulated by numerous factors, including variations in intracellular [Ca 2+ ] (13)(14)(15)(16)(17). The NA-induced phenotypes identified in the present study allowed for the following hypothesis: NA changes intracellular [Ca 2+ ], thereby obstructing cytoskeletal integrated organization, then affecting cytoskeleton-dependent intracellular transport and finally causing the accumulation of material at the perinuclear area, forming an opaque ring like structure. To confirm this hypothesis, F-actin and microtubule structures were observed with Texas Red-X phalloidin and anti-β-tubulin antibodies, respectively, under different concentrations of NA. In the PBS-treated control group, the F-actin (Fig. 2C) and microtubules (Fig. 2D) were normally patterened, as shown in the merged image in Fig. 2E. The F-actin (Fig. 2F) filaments began to disassemble, forming punctuated spots and the microtubules (Fig. 2G) exhibited weaker staining following treatment with 30 mM/1 h NA. Following incubation with 70 mM/1 h NA, the F-actin (Fig. 2I) and microtubule (Fig. 2J) cytoskeletons (merged in Fig. 2K) were completely disassembled, and the liberated microtubule residues accumulated at the distal end of the filopodia and perinuclear region (Fig. 2J). Further analysis confirmed the occurrence of microtubule disassembly with 70 mM NA in the 293T (Fig. 2L and M) and CHO-K1 cell lines ( Fig. 2N and O). These data indicated that NA dissociates the F-actin and microtubule cytoskeleton, which may affect intracellular transport in a dose-dependent manner.

Abnormal increases in the Ca 2+ concentration contribute to the disassembly of F-actin.
To further elucidate the association between changes in [Ca 2+ ] and the disassembly of the cytoskeleton, a CaCl 2 solution was added directly into the NIH3T3 cell culture media and the cytoskeleton structure was examined following 1 h of incubation. Consistent with the effect of NA demonstrated above, 40 mM CaCl 2 disrupted F-actin filaments (Fig. 3A) into punctuate G-actin   spots (Fig. 3D). However, artificial increases in [Ca 2+ ] did not affect the microtubular structure (Fig. 3E) compared with that of the control group (Fig. 3B). The results implied that the Ca 2+ wave induced by NA may be involved in the disruption of F-actin filaments, but not in the disassembly of the microtubular polymer structure.
Depolymerized microtubule subunits undergo ubiquitin-proteasome degradation. Microtubules consist of α-tubulin and β-tubulin hetero-subunits. The microtubules were labeled with anti-β tubulin antibody. Under exposure to 70 mM NA for 1 h, not only did the microtubule-stained pattern change, but also its immunofluorescent intensity decreased significantly (Fig. 2J, M and O). To confirm these results, a total amount of β tubulin was analyzed using western blot analysis. As expected, NA markedly downregulated β-tubulin at the protein level. In addition, MG132, an inhibitor of the ubiquitin-proteasome pathway, was able to reverse β-tubulin reduction (Fig. 4). However, there were no significant changes in F-actin monomer protein G-actin (data not shown).

NA blocks melanosome intracellular transport in xenopus embryos. In the cultured cells NA disrupted cytoskeletal integrity and may have inhibited intracellular trafficking.
To investigate the effect of NA on intracellular transport processes in vivo, 70 ng NA was microinjected into xenopus embryos and its effect on melanosome transport in melanocytes was observed. Melanosomes either disperse or aggregate along microtubules and F actin-filaments (18). As they are easy to observe, melanocytes represent a reliable system for investigating intracellular transport. In normal embryos, the melanosomes disperse uniformly in a dendritic type manner ( Fig. 5A and C). By contrast, in NA-microinjected embryos, melanosome transport was blocked and exhibited an aggregated disc type morphology ( Fig. 5B and D), suggesting that NA blocked intracellular transport processes in vivo.  (19). In the presence of NA and nicotinamide adenine dinucleotide phosphate (NADP), ADP-ribosyl cyclase catalyzes the synthesis of NAADP by a base exchange reaction and cAMP is a stimulator during this process (20)(21)(22). Notably, NA reduces the intracellular cAMP concentration (5). At a high concentration, NA may inhibit cAMP and thereby limit the synthesis of NAADP. The decrease of NAADP may be responsible for the first [Ca 2+ ] drop upon exposure to NA. Excessive NA may rapidly overcome the cAMP-limited step and promote the synthesis of NAADP. Therefore, a marked [Ca 2+ ] elevation was observed. Furthermore, a markedly high concentration of NA (100 mM) may completely eradicate cAMP and re-establish cAMP as a rate-limiting step. Therefore, the [Ca 2+ ] elevation curve following 100 mM NA treatment is not as steep as that following 10 mM NA treatment. In the present study, 2 mM 8-Br-cAMP (a cAMP analog) delayed and alleviated the first [Ca 2+ ] drop in response to NA, suggesting that cAMP has a key role in the changes in the [Ca 2+ ] wave induced by NA.

Discussion
It is well established that the intracellular Ca 2+ wave may modulate the cytoskeletal structure (23)(24)(25)(26)(27)(28). Although F-actins and microtubules underwent disassembly upon incubation with NA, the external addition of high concentrations of CaCl 2 only disrupted the F-actin filaments. It was hypothesized that besides the [Ca 2+ ] elevation, other pathways must also be involved in the disassembly of microtubules. The disruption of F-actin and microtubule cytoskeleton may definitely negatively effect the intracellular traffic process. In cultured cells, an opaque material accumulated around the nucleus when incubated with 70 mM NA. It appears that the minus end (nuclear region) to plus end (cell membrane region)-directed transport process was inhibited and therefore, cargo was deposited in the perinuclear region. Further evidence in the xenopus melanocyte system confirmed that NA induced an intracytic transport deficiency.
In conclusion, the present study showed that NA regulated the intracellular calcium concentration depending on its initial concentration and exposure time. High concentrations of nicotinic acid induced cytoskeletal disassembly and promoted β-tubulin degradation in a proteasome-dependent manner. The cytoskeletal disassembly may finally contribute to the disruption of the intracellular transport process. Further investigations aim to minimize the functional concentration of NA and characterize the function of NA in different biological systems, particularly in cancer cells and animal models. As the cytoskeleton is essential during cell migration and EMT, interrupting the dynamic arrangement of the cytoskeletion may break the fundamental cancerous processes of metastasis. NA provides potential for clinical use in the future. National Natural Science Foundation of China (grant nos. 81102519 to Y.S. and 81200878 to J.L.), the China Postdoctoral Science Foundation funded project (no. 2012M511914 to Y.S.), and the Chongqing Science and Technology Committee (no. cstc2012jjA0147 to Y.S.). The funders had no role in the study design, data collection and analysis, decision to publish or preparation of the manuscript.